RNA antagonist compounds for the modulation of PIK3CA expression

ABSTRACT

The invention relates to oligomeric compounds (oligomers), which target PIK3CA mRNA in a cell, leading to reduced expression of PIK3CA. Reduction of PIK3CA expression is beneficial for the treatment of certain medical disorders, such as hyperproliferative diseases (e.g., cancer). The invention provides therapeutic compositions that include the oligomers and methods for modulating the expression of PIK3CA using said oligomers, including methods of treatment.

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application Ser. No. 60/992,050, filed Dec. 3, 2007, thedisclosure of which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The invention relates to oligomeric compounds (oligomers) that targetPIK3CA mRNA in a cell, leading to reduced expression of PIK3CA. Inparticular, this invention relates to oligomeric compounds (oligomers),which target PIK3CA mRNA in a cell, leading to reduced expression ofPIK3CA. Reduction of PIK3CA expression is beneficial for a range ofmedical disorders, such as hyperproliferative diseases such as cancer.

BACKGROUND

Phospatidylinositol 3-kinase (PI3K) is a ubiquitous lipid kinaseinvolved in receptor signal transduction by tyrosine kinase receptors.PI3K comprises a large and complex family that includes 3 classes withmultiple subunits and isoforms. The class I PI3Ks are composed of a Srchomology-2 domain containing an 85 kDa regulatory subunit (p85) and a100-kDa catalytic subunit (p110), which catalyses the phosphorylation ofphosphoinositol 4-phosphate and phosphoinisitol 4,5-phosphate at theirD3 positions. The PI3K regulatory subunits include p85alpha and itstruncated splice variants p50alpha and p55alpha, as well as p85beta andp55gamma; the catalytic subunits include p110alpha, p110beta, andp110delta. The human catalytic subunit p110alpha is encoded by thePIK3CA gene, located on the human chromosome 3 [Chr 3: 180.35-180.44 Mbp] specifically [chr3:180, 349, 005-180, 435, 191 bp] (NCBI referencesequence annotation) (3q26.3), which is frequently mutated in a varietyof human cancers; PIK3CA has been shown to be mutated in 32% ofcolorectal cancers, 27% of glioblastomas, 25% of gastric cancers, 36% ofhepatocellular carcinomas, 18-40% of breast cancers, 4-12% of ovariancancers and 4% of lung cancers (Samuels et al., 2006). Most of thesemutations map to three mutational hot-spots within the PIK3CA codingsequence, which are E542K, E545K and H1047R (Kang et al., 2005).

PI3K has been indicated in a wide range of cancers, such as colorectalcarcinoma, where it is has been shown that the activation of PI3K/Akt isassociated with colorectal carcinoma and can convert differentiatedhuman gastric or colonic carcinoma cells to a less differentiated andmore malignant phenotype (Rychahou et al 2006).

The effects of PI3K on tumor growth and progression are thought to bemediated by Akt, a downstream effector of PI3K. In humans there arethree members of the Akt gene family, Akt 1, Akt 2 and Akt3. Akt is overexpressed in a number of cancers, including colon, pancreatic, ovarianand some steroid hormone-insensitive breast cancers.

Inhibitors of proteins that are involved in the PI3K/Akt signalling,which have been suggested as potential therapeutic agents, include bothsiRNAs and antisense oligonucleotides (US2006/030536A), however to datemost research in this area appears to have focused on the use of siRNAs.

WO2005/091849 describes antisense down-regulation of PI3K, however nospecific antisense oligonucleotides are disclosed.

Zhang et al., 2004 (Cancer Biology and Therapy 3:12 1283-1289) disclosessiRNAs targeting p110alpha and suggests its use in gene therapy inovarian cancer.

Rychahou et al 2006 (Annals of Surgery 243833-844) discloses siRNAcomplexes targeting p85alpha and p110alpha which were found to decreasein vitro colon cancer cell survival and to increase apoptosis in humancolon cancer cells, and decreased liver metastasis in in vivoexperiments.

Meng et al., 2006 (Cellular Signalling 18 2262-2271) discloses siRNAstargeting p110alpha for inhibiting PI3K activity in ovarian cancercells. The authors determined that inhibition of AKT1 is sufficient toaffect cell migration, invasion and proliferation.

Hsieh et al., 2004 (NAR 32 893-901) reports on the use of 148 siRNAduplexes targeting 30 genes within the PI3K pathway.

US 2005/0272682 discloses siRNA complexes targeting a phosphoinositide3-kinase (PI3K) signal transduction pathway.

In certain embodiments, the invention provides highly efficientantisense oligonucleotides which target the PI3K pathway, specificallythe PIK3CA mRNA, and in particular a new class of PIK3CA antagonistswhich have been selected based on the use of LNA chemistry, and/or bythe selection of particularly effective target sites on the PIK3CA mRNA.

SUMMARY OF INVENTION

The invention provides an oligomer of 10-50 monomers, such as 10-30monomers which comprises a first region of 10-30 monomers, wherein thesequence of the first region is at least 80% (e.g., 85%, 90%, 95%, 98%,or 99%) identical to the reverse complement of a target region of anucleic acid which encodes a mammalian PIK3CA, such as a mammalianPIK3CA gene or mRNA, such as a nucleic acid have the sequence set forthin SEQ ID NO: 1 or naturally occurring variants thereof.

The invention provides for a conjugate comprising the oligomer accordingto the invention, and at least one non-nucleoside or non-polynucleotidemoiety covalently attached to the oligomer.

The invention provides for a pharmaceutical composition comprising theoligomer or the conjugate according to the invention, and apharmaceutically acceptable diluent, carrier, salt or adjuvant.

The invention provides for the oligomer or the conjugate according tothe invention, for use as a medicament, such as for the treatment ofhyperproliferative diseases, such as cancer.

The invention provides for the use of an oligomer or conjugate thereofaccording to the invention, for the manufacture of a medicament for thetreatment of hyperproliferative diseases such as cancer.

The invention provides for a method of treating a hyperproliferativedisease such as cancer, the method comprising administering an effectiveamount of an oligomer, a conjugate or a pharmaceutical compositionaccording to the invention, to a patient suffering from, or susceptibleto, said the hyperproliferative disease.

The invention provides for a method of inhibiting PIK3CA in a cell whichis expressing PIK3CA, the method comprising contacting the cell in vitroor in vivo with an effective amount of an oligomer, or a conjugateaccording to the invention to effect the inhibition of PIK3CA expressionin the cell.

The invention further provides for an oligomer according to theinvention, for use in medicine.

The invention further provides for an oligomer according to theinvention, for use for the treatment of one or more of the diseasesreferred to herein, such as a disease selected from the group consistingof hyperproliferative diseases, such as cancer.

Also disclosed are methods of treating a non-human animal or a human,suspected of having or being susceptible to a hyperproliferativedisease, such as cancer and/or other hyperproliferative diseases,associated with the expression, or over-expression, of PIK3CA, byadministering to the non-human animal or human a therapeutically orprophylactically effective amount of an oligomer, conjugate orcomposition of the invention. Further, methods of using oligomers forthe inhibition of expression of PIK3CA, and for treatment of diseasesassociated with PIK3CA (e.g., PI3K) are provided.

The invention provides for a method for treating a disease selected fromthe group consisting of hyperproliferative diseases such as cancer; themethod comprises administering an effective amount of an oligomer, aconjugate, or a pharmaceutical composition according to the invention toa patient in need thereof.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Oligonucleotides presented in Table 5 were evaluated for theirpotential to knock down the PIK3CA mRNA at concentrations of 0.8 nM, 4nM and 20 nM in MCF7 cells 24 hours after transfection using Real-timePCR. All results were normalised to GAPDH and inhibition of PIK3CA mRNAis shown as percent of mock-transfected control. Results shown are theaverage of results from three independent experiments.

FIG. 2. Oligonucleotides presented in Table 5 were evaluated for theirpotential to knock down the PIK3CA mRNA at concentrations of 0.8 nM, 4nM and 20 nM in PC3 cells 24 hours after transfection using Real-timePCR. All results were normalised to GAPDH and inhibition of PIK3CA mRNAis shown as percent of mock-transfected control. Results shown are theaverage of results from three independent experiments.

FIG. 3. Sequence alignment of the human PIK3CA mRNA sequence, GenBankAccession number NM_(—)006218 (SEQ ID NO: 1) and the mouse PIK3CA mRNAsequence, Gen Bank Accession number NM_(—)008839(SEQ ID NO: 106).Consensus sequence disclosed as SEQ ID NO: 164.

FIG. 4. Location of target regions on the human PIK3CA mRNA sequence(GenBank Accession number NM_(—)006218) (SEQ ID NO: 1). Positions markedin grey are mutation hot-spots- 1781, 1790 and 3297.

FIG. 5. SEQ ID NO: 1 Homo sapiens phosphoinositide-3-kinase, catalytic,alpha polypeptide (PIK3CA) mRNA, GenBank Accession number NM_(—)006218,3724 bp.

FIG. 6. SEQ ID NO 105. Homo sapiens phosphoinositide-3-kinase catalytic,alpha polypeptide protein sequence (PIK3CA) GenBank Accession numberNP_(—)006209.

FIG. 7. SEQ ID NO 106. Mus musculus phosphatidylinositol 3-kinase,catalytic, alpha polypeptide (PIK3CA), mRNA. GenBank Accession numberNM_(—)008839.

FIG. 8. SEQ ID NO 107. Mus musculus phosphoinositide-3-kinase catalytic,alpha polypeptide protein sequence (PIK3CA) GenBank Accession numberNP_(—)032865.

FIG. 9. SEQ ID NO 108. Macaca mulatta phosphatidylinositol 3-kinase,catalytic, alpha polypeptide (PIK3CA), mRNA. GenBank Accession numberXM_(—)001109162.

FIG. 10. SEQ ID NO 109. Macaca mulatta phosphoinositide-3-kinasecatalytic, alpha polypeptide protein sequence (PIK3CA) GenBank Accessionnumber XP_(—)001109162.

FIG. 11. Cell proliferation assay (MTS assay) in MCF7 cells. Fourindependent experiments were performed. The results from the experimentbest representing the average activity of each oligomer are shown.

FIG. 12. Cell proliferation assay (MTS) in PC3 cells. Two independentexperiments were performed. The results from the experiment bestrepresenting the average activity of each oligomer are shown.

FIG. 13. Caspase 3/7 activity in PC3 cells after transfection withPIK3CA oligonucleotides. Data are expressed as fold induction comparedto mock.

FIG. 14. Cell proliferation assay (MTS) in HCT116 cells.

FIG. 15. Caspase 3/7 activity in HCT116 cells after transfection withPIK3CA oligomers. Data are expressed as fold induction compared to mock.

FIG. 16. Plasma stability of PIK3CA oligonucleotides. The LNAoligonucleotides were incubated with mouse plasma at 37° C. and aliquotswere taken at 0, 24, 48 and 120 h. The results were visualized by gelelectrophoresis using an SDS-PAGE gel.

FIG. 17. Tm determination of PIK3CA oligonucleotides hybridised to atarget region of a target nucleic acid. Bold, uppercase letters with asuperscript “o” to the right representβ-D-oxy LNA monomers. MCrepresents LNA monomers with 5-methylcytosine bases. Subscript “s”represents a phosphorothioate linkage. Lowercase letters represent DNAmonomers.

FIG. 18. Down-regulation of PIK3CA protein and pAkt in A549 cells.

FIG. 19. Down-regulation of PIK3CA protein and pAkt in 15PC3 cells.

FIG. 20. Analysis of knock down of PIK3CA mRNA in liver.

FIG. 21. In vivo knock down of PIK3CA mRNA in mouse liver. Data areexpressed as % down-regulation compared to saline (100%)±stdev. Therewere 5 animals in each group.

FIG. 22. The most potent oligonucleotides were evaluated for theirpotential to knock down the PIK3CA mRNA at concentrations of 0.04 nM,0.2 nM, 0.8 nM, 4 nM, 10 nM and 20 nM in PC3 cells 24 hours aftertransfection using Real-time PCR. All results were normalised to GAPDHand inhibition of PIK3CA mRNA is shown as percent of mock-transfectedcontrol. Results shown are the average of two independent experiments.

FIG. 23. The most potent oligonucleotides were evaluated for theirpotential to knock down the PIK3CA mRNA at concentrations of 0.04 nM,0.2 nM, 0.8 nM, 4 nM, 10 nM and 20 nM in MCF7 cells 24 hours aftertransfection using Real-time PCR. All results were normalised to GAPDHand inhibition of PIK3CA mRNA is shown as percent of mock-transfectedcontrol. Results shown are the average of two independent experiments.

DETAILED DESCRIPTION OF INVENTION

The Oligomer

The invention employs oligomeric compounds (referred herein asoligomers), for use in modulating the function of nucleic acid moleculesencoding mammalian PIK3CA, such as the PIK3CA nucleic acid having thesequence shown in SEQ ID NO.: 1, and naturally occurring variants ofsuch nucleic acid molecules encoding mammalian PIK3CA. The term“oligomer” in the context of the present invention, refers to a moleculeformed by covalent linkage of two or more monomers (i.e. anoligonucleotide). In some embodiments, the oligomer consists of orcomprises from 10-30 monomers.

The term “monomer” includes both nucleosides and deoxynucleosides(collectively, “nucleosides”) that occur naturally in nucleic acids andthat do not contain either modified sugars or modified nucleobases,i.e., compounds in which a ribose sugar or deoxyribose sugar iscovalently bonded to a naturally-occurring, unmodified nucleobase (base)moiety (i.e., the purine and pyrimidine heterocycles adenine, guanine,cytosine, thymine or uracil) and “nucleoside analogues,” which arenucleosides that either do occur naturally in nucleic acids or do notoccur naturally in nucleic acids, wherein either the sugar moiety isother than a ribose or a deoxyribose sugar (such as bicyclic sugars or2′ modified sugars, such as 2′ substituted sugars), or the base moietyis modified (e.g., 5-methylcytosine), or both.

An “RNA monomer” is a nucleoside containing a ribose sugar and anunmodified nucleobase.

A “DNA monomer” is a nucleoside containing a deoxyribose sugar and anunmodified nucleobase.

A “Locked Nucleic Acid monomer,” “locked monomer,” or “LNA monomer” is anucleoside analogue having a bicyclic sugar, as further described hereinbelow.

The terms “corresponding nucleoside analogue” and “correspondingnucleoside” indicate that the base moiety in the nucleoside analogue andthe base moiety in the nucleoside are identical. For example, when the“nucleoside” contains a 2-deoxyribose sugar linked to an adenine, the“corresponding nucleoside analogue” contains, for example, a modifiedsugar linked to an adenine base moiety.

The terms “oligomer,” “oligomeric compound,” and “oligonucleotide” areused interchangeably in the context of the invention, and refer to amolecule formed by covalent linkage of two or more contiguous monomersby, for example, a phosphate group (forming a phosphodiester linkagebetween nucleosides) or a phosphorothioate group (forming aphosphorothioate linkage between nucleosides). The oligomer consists of,or comprises, 10-50 monomers, such as 10-30 monomers.

In some embodiments, an oligomer comprises nucleosides, or nucleosideanalogues, or mixtures thereof as referred to herein. An “LNA oligomer”or “LNA oligonucleotide” refers to an oligonucleotide containing one ormore LNA monomers.

The terms “corresponding nucleoside analogue” and “correspondingnucleoside” indicate that the base moiety in the nucleoside analogue andthe base moiety in the nucleoside are identical. For example, when the“nucleoside” contains a 2-deoxyribose sugar linked to an adenine, the“corresponding nucleoside analogue” contains, for example, a modifiedsugar linked to an adenine base moiety.

In some embodiments, an oligomer comprises nucleosides, or nucleosideanalogues, or mixtures thereof as referred to herein. An “LNA oligomer”or “LNA oligonucleotide” refers to an oligonucleotide containing one ormore LNA monomers.

Nucleoside analogues that are optionally included within oligomers mayfunction similarly to corresponding nucleosides, or may have specificimproved functions. Oligomers wherein some or all of the monomers arenucleoside analogues are often preferred over native forms because ofseveral desirable properties of such oligomers, such as the ability topenetrate a cell membrane, good resistance to extra- and/orintracellular nucleases and high affinity and specificity for thenucleic acid target. LNA monomers are particularly preferred, forexample, for conferring several of the above-mentioned properties.

In various embodiments, one or more nucleoside analogues present withinthe oligomer are “silent” or “equivalent” in function to thecorresponding natural nucleoside, i.e., have no functional effect on theway the oligomer functions to inhibit target gene expression. Such“equivalent” nucleoside analogues are nevertheless useful if, forexample, they are easier or cheaper to manufacture, or are more stableunder storage or manufacturing conditions, or can incorporate a tag orlabel. Typically, however, the analogues will have a functional effecton the way in which the oligomer functions to inhibit expression; forexample, by producing increased binding affinity to the target region ofthe target nucleic acid and/or increased resistance to intracellularnucleases and/or increased ease of transport into the cell.

Thus, in various embodiments, oligomers according to the inventioncomprise nucleoside monomers and at least one nucleoside analoguemonomer, such as an LNA monomer, or other nucleoside analogue monomers.

The term “at least one” comprises the integers larger than or equal to1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 and so forth. In various embodiments, such as when referringto the nucleic acid or protein targets of the compounds of theinvention, the term “at least one” includes the terms “at least two” and“at least three” and “at least four.” Likewise, in some embodiments, theterm “at least two” comprises the terms “at least three” and “at leastfour.”

In some embodiments, the oligomer comprises or consists of 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30contiguous monomers.

In some embodiments, the oligomer comprises or consists of 10-22contiguous monomers, such as 12-18 contiguous monomers, such as 13-17 or12-16 contiguous monomers, such as 13, 14, 15, 16 contiguous monomers.

In certain embodiments, the oligomer comprises or consists of 10, 11,12, 13, or 14 contiguous monomers.

In various embodiments, the oligomer according to the invention consistsof no more than 22 monomers, such as no more than 20 monomers, such asno more than 18 monomers, such as 15, 16 or 17 monomers. In someembodiments, the oligomer of the invention comprises less than 20monomers.

In various embodiments, the compounds of the invention do not compriseRNA monomers.

In various embodiments, the compounds according to the invention arelinear molecules or are linear as synthesised. The oligomer, in suchembodiments, is a single stranded molecule, and typically does notcomprise short regions of, for example, at least 3, 4 or 5 contiguousmonomers, which are complementary to another region within the sameoligomer such that the oligomer forms an internal duplex. In someembodiments, the oligomer is essentially not double stranded, i.e., isnot a siRNA.

In some embodiments, the oligomer of the invention consists of acontiguous stretch of monomers, the sequence of which is identified by aSEQ ID NO disclosed herein (see, e.g., Tables 2-5). In otherembodiments, the oligomer comprises a first region, the regionconsisting of a contiguous stretch of monomers, and one or moreadditional regions which consist of at least one additional monomer. Insome embodiments, the sequence of the first region is identified by aSEQ ID NO disclosed herein.

Gapmer Design

Typically, the oligomer of the invention is a gapmer.

A “gapmer” is an oligomer which comprises a contiguous stretch ofmonomers capable of recruiting an RNAse (e.g., such as RNAseH) asfurther described herein below, such as a region of at least 6 or 7 DNAmonomers, referred to herein as region B, wherein region B is flankedboth on its 5′ and 3′ ends by regions respectively referred to asregions A and C, each of regions A and C comprising or consisting ofnucleoside analogues, such as affinity-enhancing nucleoside analogues,such as 1-6 nucleoside analogues.

Typically, the gapmer comprises regions, from 5′ to 3′, A-B-C, oroptionally A-B-C-D or D-A-B-C, wherein: region A consists of orcomprises at least one nucleoside analogue, such as at least one LNAmonomer, such as 1-6 nucleoside analogues, such as LNA monomers, andregion B consists of or comprises at least five contiguous monomerswhich are capable of recruiting RNAse (when formed in a duplex with acomplementary target region of the target RNA molecule, such as the mRNAtarget), such as DNA monomers; region C consists of or comprises atleast one nucleoside analogue, such as at least one LNA monomer, such as1-6 nucleoside analogues, such as LNA monomers; and region D, whenpresent, consists of or comprises 1, 2 or 3 monomers, such as DNAmonomers.

In various embodiments, region A consists of 1, 2, 3, 4, 5 or 6nucleoside analogues, such as LNA monomers, such as 2-5 nucleosideanalogues, such as 2-5 LNA monomers, such as 3 or 4 nucleosideanalogues, such as 3 or 4 LNA monomers; and/or region C consists of 1,2, 3, 4, 5 or 6 nucleoside analogues, such as LNA monomers, such as 2-5nucleoside analogues, such as 2-5 LNA monomers, such as 3 or 4nucleoside analogues, such as 3 or 4 LNA monomers.

In certain embodiments, region B consists of or comprises 5, 6, 7, 8, 9,10, 11 or 12 contiguous monomers which are capable of recruiting RNAse,or 6-10, or 7-9, such as 8 contiguous monomers which are capable ofrecruiting RNAse. In certain embodiments, region B consists of orcomprises at least one DNA monomer, such as 1-12 DNA monomers,preferably 4-12 DNA monomers, more preferably 6-10 DNA monomers, such as7-10 DNA monomers, most preferably 8, 9 or 10 DNA monomers.

In various embodiments, region A consists of 3 or 4 nucleosideanalogues, such as LNA monomers, region B consists of 7, 8, 9 or 10 DNAmonomers, and region C consists of 3 or 4 nucleoside analogues, such asLNA monomers. Such designs include (A-B-C) 3-10-3, 3-10-4, 4-10-3,3-9-3, 3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3, and mayfurther include region D, which may have one or 2 monomers, such as DNAmonomers.

Further gapmer designs are disclosed in WO2004/046160, which is herebyincorporated by reference.

US provisional application, 60/977,409, hereby incorporated byreference, refers to ‘shortmer’ gapmer oligomers. In some embodiments,oligomers presented here may be such shortmer gapmers.

In certain embodiments, the oligomer consists of 10, 11, 12, 13 or 14contiguous monomers, wherein the regions of the oligomer have thepattern (5′-3′), A-B-C, or optionally A-B-C-D or D-A-B-C, wherein:region A consists of 1, 2 or 3 nucleoside analogue monomers, such as LNAmonomers; region B consists of 7, 8 or 9 contiguous monomers which arecapable of recruiting RNAse when formed in a duplex with a complementaryRNA molecule (such as a mRNA target); and region C consists of 1, 2 or 3nucleoside analogue monomers, such as LNA monomers. When present, regionD consists of a single DNA monomer.

In certain embodiments, region A consists of 1 LNA monomer. In certainembodiments, region A consists of 2 LNA monomers. In certainembodiments, region A consists of 3 LNA monomers. In certainembodiments, region C consists of 1 LNA monomer. In certain embodiments,region C consists of 2 LNA monomers. In certain embodiments, region Cconsists of 3 LNA monomers. In certain embodiments, region B consists of7 nucleoside monomers. In certain embodiments, region B consists of 8nucleoside monomers. In certain embodiments, region B consists of 9nucleoside monomers. In certain embodiments, region B comprises 1-9 DNAmonomers, such as 2, 3, 4, 5, 6, 7 or 8 DNA monomers. In certainembodiments, region B consists of DNA monomers. In certain embodiments,region B comprises at least one LNA monomer which is in the alpha-Lconfiguration, such as 2, 3, 4, 5, 6, 7, 8 or 9 LNA monomers in thealpha-L-configuration. In certain embodiments, region B comprises atleast one alpha-L-oxy LNA monomer. In certain embodiments, all the LNAmonomers in region B that are in the alpha-L-configuration arealpha-L-oxy LNA units. In certain embodiments, the number of monomerspresent in the A-B-C regions are selected from the group consisting of(nucleoside analogue monomers—region B—nucleoside analogue monomers):1-8-1, 1-8-2, 2-8-1, 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2, 1-8-4,2-8-4, or; 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2, 1-9-3, 3-9-1,4-9-1, 1-9-4, or; 1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, 3-10-1. Incertain embodiments, the number of monomers present in the A-B-C regionsof the oligomer of the invention is selected from the group consistingof: 2-7-1, 1-7-2, 2-7-2, 3-7-3, 2-7-3, 3-7-2, 3-7-4, and 4-7-3. Incertain embodiments, each of regions A and C consists of two LNAmonomers, and region B consists of 8 or 9 nucleoside monomers,preferably DNA monomers.

In various embodiments, other gapmer designs include those where regionsA and/or C consists of 3, 4, 5 or 6 nucleoside analogues, such asmonomers containing a 2′-O-methoxyethyl-ribose sugar (2′-MOE) ormonomers containing a 2′-fluoro-deoxyribose sugar, and region B consistsof 8, 9, 10, 11 or 12 nucleosides, such as DNA monomers, where regionsA-B-C have 5-10-5 or 4-12-4 monomers. Further gapmer designs aredisclosed in WO 2007/146511A2, hereby incorporated by reference.

Internucleoside Linkages

The monomers of the oligomers described herein are coupled together vialinkage groups. Suitably, each monomer is linked to the 3′ adjacentmonomer via a linkage group.

The terms “linkage group” or “internucleoside linkage” means a groupcapable of covalently coupling together two contiguous monomers.Specific and preferred examples include phosphate groups (forming aphosphodiester between adjacent nucleoside monomers) andphosphorothioate groups (forming a phosphorothioate linkage betweenadjacent nucleoside monomers).

Suitable linkage groups include those listed in PCT/DK2006/000512, forexample in the first paragraph of page 34 of PCT/DK2006/000512 (herebyincorporated by reference).

It is, in various embodiments, preferred to modify the linkage groupfrom its normal phosphodiester to one that is more resistant to nucleaseattack, such as phosphorothioate or boranophosphate—these two beingcleavable by RNaseH, thereby permitting RNase-mediated antisenseinhibition of expression of the target gene.

In some embodiments, suitable sulphur (S) containing linkage groups asprovided herein are preferred. In various embodiments, phosphorothioatelinkage groups are preferred, particularly for the gap region (B) ofgapmers. In certain embodiments, phosphorothioate linkages are used tolink together monomers in the flanking regions (A and C). In variousembodiments, phosphorothioate linkages are used for linking regions A orC to region D, and for linking together monomers within region D.

In various embodiments, regions A, B and C, comprise linkage groupsother than phosphorothioate, such as phosphodiester linkages,particularly, for instance when the use of nucleoside analogues protectsthe linkage groups within regions A and C from endo-nucleasedegradation—such as when regions A and C comprise LNA monomers.

In various embodiments, adjacent monomers of the oligomer are linked toeach other by means of phosphorothioate groups.

It is recognised that the inclusion of phosphodiester linkages, such asone or two linkages, into an oligomer with a phosphorothioate backbone,particularly with phosphorothioate linkage groups between or adjacent tonucleoside analogue monomers (typically in region A and/or C), canmodify the bioavailability and/or bio-distribution of an oligomer—seeWO2008/053314, hereby incorporated by reference.

In some embodiments, such as the embodiments referred to above, wheresuitable and not specifically indicated, all remaining linkage groupsare either phosphodiester or phosphorothioate, or a mixture thereof.

In some embodiments all the internucleoside linkage groups arephosphorothioate.

When referring to specific gapmer oligonucleotide sequences, such asthose provided herein, it will be understood that, in variousembodiments, when the linkages are phosphorothioate linkages,alternative linkages, such as those disclosed herein may be used, forexample phosphate (phosphodiester) linkages may be used, particularlyfor linkages between nucleoside analogues, such as LNA monomers.Likewise, in various embodiments, when referring to specific gapmeroligonucleotide sequences, such as those provided herein, when one ormore monomers in region C comprises a 5-methylcytosine base, othermonomers in that region may contain unmodified cytosine bases.

Target Nucleic Acid

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein, and are defined as a molecule formed by covalent linkage of twoor more monomers, as above-described. Including 2 or more monomers,“nucleic acids” may be of any length, and the term is generic to“oligomers”, which have the lengths described herein. The terms “nucleicacid” and “polynucleotide” include single-stranded, double-stranded,partially double-stranded, and circular molecules.

The term “target nucleic acid”, as used herein, refers to DNA or RNA(e.g., mRNA or pre-mRNA) encoding a mammalian PIK3CA polypeptide, suchas human PIK3CA, such as the nucleic acid having the sequence shown inSEQ ID NO: 1, and naturally occurring allelic variants of such nucleicacids. In certain embodiments, the target nucleic acid encodes a mousePIK3CA polypeptide. In some embodiments, target nucleic acid refers toDNA or RNA that encodes a mammalian PIK3CA polypeptide and DNA or RNAthat encodes a mammalian beta-catenin polypeptide. The oligomers of theinvention are typically capable of hybridising to the target nucleicacid(s).

The term “naturally occurring variant thereof” refers to variants of thePIK3CA polypeptide or nucleic acid sequence which exist naturally withinthe defined taxonomic group, such as mammalian, such as mouse, monkey,and preferably human PIK3CA. Typically, when referring to “naturallyoccurring variants” of a polynucleotide the term also encompasses anyallelic variant of the PIK3CA encoding genomic DNA which is found at theChromosome 3 [Chr 3: 180.35-180.44 M bp] specifically [chr3:180, 349,005-180, 435, 191 bp] (NCBI reference sequence annotation) (3q26.3) bychromosomal translocation or duplication, and the RNA, such as mRNA,derived therefrom. “Naturally occurring variants” may also includevariants derived from alternative splicing of the PIK3CA mRNA. Whenreferenced to a specific polypeptide sequence, e.g., the term alsoincludes naturally occurring forms of the protein which may therefore beprocessed, e.g. by co- or post-translational modifications, such assignal peptide cleavage, proteolytic cleavage, glycosylation, etc. Incertain embodiments, variant target nucleic acids, have at least 60%,more preferably at least 70%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 95% sequence homology (also, identity) to wild-type PIK3CA.Typically, an oligomer of the invention corresponding to (i.e., that iscomplementary to and binds to) a target region that contains one or moremutations compared to the wild-type PIK3CA sequence is still capable ofdown-regulating PIK3CA expression. In certain embodiments, the mutationis “silent” (i.e. is not associated with an altered phenotype ordisease). In various embodiments, the mutation is “functional” (i.e.,disease-associated). In various embodiments, the naturally occurringvariant is an allelic variant.

Particular variants of PIK3CA, which in some embodiments are targeted bythe oligomers of the invention, contain spontaneous point mutationswhich are associated with hyperproliferative diseases such as cancer. Incertain embodiments, such point mutations are those which result inamino acid substitutions at positions E542, E545 or H1047, such as pointmutations which result in the amino acid substitutions E542K, E545K orH1047R. By designing oligomers which target (e.g., are fullycomplementary to) one or more target regions of PIK3CA mRNA whichcomprises such a point mutation, in certain embodiments, oligomers ofthe invention preferentially down-regulate (i.e., by inhibiting) theexpression of such variant forms of PIK3CA mRNA. In certain embodiments,the oligomers that target these variant forms of PIK3CA selectivelydown-regulate one or more variant PIK3CA mRNAs (e.g., those havingmutations at positions E542, E545 or H1047) that are associated with acancer phenotype.

In certain embodiments, the PIK3CA gene or mRNA target nucleic acidcomprises a target region containing a single base substitution atposition 1781, 1790 or 3297 of SEQ ID NO 1. In such embodiments, thenucleobase of the monomer at position 1781 of SEQ ID NO: 1 is other thanG (e.g., A, C or T/U) the nucleobase of the monomer at position 1790 isother than G, or the nucleobase of the monomer at position 3297 is otherthan A.

In certain embodiments, the oligomer according to the inventioncomprises a sequence that is fully complementary to a target region thatincludes a point mutation at position 1781 of SEQ ID NO: 1. In theseembodiments, the nucleobase of the monomer in the oligomer thatbase-pairs with the nucleobase of the monomer at position 1781 of SEQ IDNO: 1 is not C (e.g., the oligomer is not fully complementary to thewild-type target region. In other embodiments, the oligomer of theinvention comprises one mismatch when compared to the best alignedtarget region of SEQ ID NO: 1 that includes a point mutation at position1781 of SEQ ID NO: 1.

In various embodiments, the oligomer according to the inventioncomprises a sequence that is fully complementary to a target region thatincludes a point mutation at position 1790 of SEQ ID NO 1. In theseembodiments, the nucleobase of the monomer of the oligomer thatbase-pairs with the nucleobase of the monomer at position 1790 in SEQ IDNO: 1 is not C (e.g., the oligomer is not fully complementary to thewild-type target region. In other embodiments, the oligomer comprisesone mismatch when compared to the best-aligned target region of SEQ IDNO: 1 that includes a point mutation at position 1790 of SEQ ID NO: 1.

In various embodiments, the oligomer according to the inventioncomprises a sequence that is fully complementary to a target region thatincludes a point mutation at position 3297 of SEQ ID NO: 1, wherein thenucleobase of the monomer of the oligomer which base-pairs with thenucleobase of the monomer at position 3297 is not T (e.g., the oligomeris not fully complementary to the wild-type target region). In otherembodiments, the oligomer of the invention comprises one mismatch whencompared to the best-aligned target region of SEQ ID NO: 1 that includesa point mutation at position 3297.

In some embodiments, for example when used in research or diagnostics,the “target nucleic acid” is a cDNA or a synthetic oligonucleotidederived from the above DNA or RNA nucleic acid targets. It will berecognised that the nucleic acid having the sequence as set forth in SEQID NO: 1 is a cDNA, and as such, has the same base sequence as themature mRNA target, although uracil (U) bases are replaced by thymidine(T) bases in the cDNA.

In certain embodiments, oligomers described herein bind to a region ofthe target nucleic acid (the “target region”) by either Watson-Crickbase pairing, Hoogsteen hydrogen bonding, or reversed Hoogsteen hydrogenbonding, between the monomers of the oligomer and monomers of the targetnucleic acid. Such binding is also referred to as “hybridisation.”Unless otherwise indicated, binding is by Watson-Crick pairing ofcomplementary bases (i.e., adenine with thymine (DNA) or uracil (RNA),and guanine with cytosine), and the oligomer binds to the target regionbecause the sequence of the oligomer is identical to, orpartially-identical to, the sequence of the reverse complement of thetarget region; for purposes herein, the oligomer is said to be“complementary” or “partially complementary” to the target region, andthe percentage of “complementarity” of the oligomer sequence to that ofthe target region is the percentage “identity” to the reverse complementof the sequence of the target region.

Unless otherwise made clear by context, the “target region” herein willbe the region of the target nucleic acid having the sequence that bestaligns with the reverse complement of the sequence of the specifiedoligomer (or region thereof), using the alignment program and parametersdescribed herein below.

In determining the degree of “complementarity” between oligomers of theinvention (or regions thereof) and the target region of the nucleic acidwhich encodes mammalian PIK3CA, such as those disclosed herein, thedegree of “complementarity” (also, “homology”) is expressed as thepercentage identity between the sequence of the oligomer (or regionthereof) and the reverse complement of the sequence of the target regionthat best aligns therewith. The percentage is calculated by counting thenumber of aligned bases that are identical as between the 2 sequences,dividing by the total number of contiguous monomers in the oligomer, andmultiplying by 100. In such a comparison, if gaps exist, it ispreferable that such gaps are merely mismatches rather than areas wherethe number of monomers within the gap differs between the oligomer ofthe invention and the target region.

Amino acid and polynucleotide alignments, percentage sequence identity,and degree of complementarity may be determined for purposes of theinvention using the ClustalW algorithm using standard settings: Method:EMBOSS::water (local): Gap Open=10.0, Gap extend=0.5, using Blosum 62(protein), or DNAfull for nucleoside/nucleobase sequences.

As will be understood, depending on context, “mismatch” refers to anon-identity in sequence (as, for example, between the nucleobasesequence of an oligomer and the reverse complement of the target regionto which it binds; as for example, between the base sequence of twoaligned PIK3CA encoding nucleic acids), or to noncomplementarity insequence (as, for example, between an oligomer and the target region towhich binds).

Suitably the oligomer of the invention or conjugate thereof is capableof down-regulating (i.e., by inhibiting) expression of the PIK3CA gene.In various embodiments, the oligomer of the invention can effect theinhibition of PIK3CA, typically in a mammalian cell, such as a humancell. In certain embodiments, the oligomers of the invention orconjugates thereof bind to the target nucleic acid and effect inhibitionof expression of at least 10% or 20% compared to the expression level ofPIK3CA in a cell immediately prior to dosing of the oligomer, morepreferably at least a 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%inhibition compared to the expression level of PIK3CA in a cellimmediately prior to dosing of the oligomer. In some embodiments, suchinhibition is seen when using from about 0.04 nM to about 25 nM, such asfrom about 0.8 nM to about 20 nM concentration of the oligomer orconjugate.

In various embodiments, the inhibition of expression is less than 100%(i.e., less than complete inhibition of expression), such as less than98% inhibition, less than 95% inhibition, less than 90% inhibition, lessthan 80% inhibition, such as less than 70% inhibition. In variousembodiments, modulation of gene expression level can be determined bymeasuring protein levels, e.g. by methods such as SDS-PAGE followed bywestern blotting using suitable antibodies raised against the targetprotein. Alternatively, in certain embodiments, modulation of PIK3CAexpression levels can be determined by measuring levels of mRNA, e.g. bynorthern blotting or quantitative RT-PCR. When measuring via mRNAlevels, the level of down-regulation when using an appropriate dosage,such as from about 0.04 nM to about 25 nM, such as from about 0.8 nM toabout 20 nM concentration, is, in some embodiments, typically to a levelof 10-20% the expression levels in the absence of the compound orconjugate of the invention.

The invention therefore provides a method of down-regulating (e.g., byinhibiting) the expression of PIK3CA protein and/or mRNA in a cell whichis expressing PIK3CA protein and/or mRNA, the method comprisingcontacting the cell with an effective amount of an oligomer or conjugateaccording to the invention to down-regulate or inhibit the expression ofPIK3CA protein and/or mRNA in the cell. Suitably, the cell is amammalian cell such as a human cell. The administration may occur, insome embodiments, in vitro. The administration may occur, in someembodiments, in vivo.

Alternatively, in certain embodiments, the invention provides for amethod of inhibiting PIK3CA and beta-catenin in a cell which isexpressing both PIK3CA and beta-catenin, the method comprisingcontacting the cell in vitro or in vivo with an effective amount of anoligomer, or a conjugate according to the invention to effect theinhibition of PIK3CA and beta-catenin expression in the cell. Suitably,the oligomer which is capable of inhibiting or down-regulating bothPIK3CA and beta-catenin in a cell has significant identity to thereverse complement of a target region of a PIK3CA nucleic acid and tothe reverse complement of a target region of a beta-catenin nucleicacid, such as an oligomer with the sequence of nucleobases set forth inSEQ ID NO: 82.

Oligomer Sequences

In certain embodiments, the oligomers of the invention have sequencesthat are identical to a sequence selected from the group consisting ofSEQ ID NOs: 2-16, 17-28, 110-124, 125-136, 149-159 and 160. In variousembodiments, the oligomers of the invention have base sequences that areselected from the group consisting of SEQ ID NOs: 29-55 and 56-148. Invarious embodiments, the oligomer has the sequence of SEQ ID NO: 67 orSEQ ID NO: 77. Further provided are target nucleic acids (e.g., DNA ormRNA encoding AR) that contain target regions that are complementary orpartially-complementary to one or more of the oligomers of theinvention. In certain embodiments, the oligomers bind to variants ofPIK3CA target regions, such as allelic variants. In some embodiments, avariant of PIK3CA target region has at least 60%, more preferably atleast 70%, more preferably at least 80%, more preferably at least 85%,more preferably at least 90%, more preferably at least 91%, at least92%, at least 93%, at least 94%, at least 95% sequence identity to thetarget region in wild-type PIK3CA. Thus, in other embodiments, theoligomers of the invention have sequences that differ in 1, 2 or 3 baseswhen compared to a sequence selected from the group consisting of SEQ IDNOs: 2-16, 17-28, 110-124, 125-136, 149-159 and 160. Typically, anoligomer of the invention that binds to a variant of a PIK3CA targetregion is capable of inhibiting (e.g., by down-regulating) PIK3CA.

In other embodiments, oligomers of the invention are LNA oligomers, forexample, those oligomers having the sequences shown in SEQ ID NOs:29-104 and 137-148. In various embodiments, the oligomers of theinvention are potent inhibitors of PIK3CA mRNA and protein expression.In various embodiments, oligomers of the invention are LNA oligomershaving the sequences of SEQ ID NO: 67 or SEQ ID NO: 77.

In various embodiments, the oligomer comprises or consists of a regionhaving a base sequence which is identical or partially identical to thesequence of the reverse complement of a target region in SEQ ID NO: 1.In various embodiments, the oligomer comprises or consists of a regionhaving a sequence selected from the group consisting of SEQ ID NOS:2-16, 17-28, 110-124, 125-136, 149-159 and 160.

In certain embodiments, the oligomer comprises or consists of a regionhaving a base sequence which is fully complementary (perfectlycomplementary) to a target region of a nucleic acid which encodes amammalian PIK3CA.

However, in some embodiments, the oligomer includes 1, 2, 3, or 4 (ormore) mismatches as compared to the best-aligned target region of aPIK3CA target nucleic acid, and still sufficiently binds to the targetregion to effect inhibition of PIK3CA mRNA or protein expression. Thedestabilizing effect of mismatches on Watson-Crick hydrogen-bondedduplex may, for example, be compensated by increased length of theoligomer and/or an increased number of nucleoside analogues, such as LNAmonomers, present within the oligomer.

In various embodiments, the oligomer base sequence comprises no morethan 3, such as no more than 2 mismatches compared to the base sequenceof the best-aligned target region of, for example, a target nucleic acidwhich encodes a mammalian PIK3CA.

In some embodiments, the oligomer base sequence comprises no more than asingle mismatch when compared to the base sequence of the best-alignedtarget region of a nucleic acid which encodes a mammalian PIK3CA.

In various embodiments, the base sequence of the oligomer of theinvention, or of a first region thereof, is preferably at least 80%identical to a base sequence selected from the group consisting of SEQID NOS: 2-16, 17-28, 110-124, 125-136, 149-159 and 160, such as at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96% identical, such as 100% identical.

In certain embodiments, the base sequence of the oligomer of theinvention or of a first region thereof is at least 80% identical to thebase sequence of the reverse complement of a target region present inSEQ ID NO: 1, such as at least 85%, at least 90%, at least 91%, at least92% at least 93%, at least 94%, at least 95%, at least 96% identical, atleast 97% identical, at least 98% identical, at least 99% identical,such as 100% identical.

In various embodiments, the base sequence of the oligomer of theinvention, or of a first region thereof, is preferably at least 80%complementary to a target region of SEQ ID NO: 1, such as at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96% complementary, at least 97% complementary, atleast 98% complementary, at least 99% complementary, such as 100%complementary (perfectly complementary).

In various embodiments, the sequence of the oligomer (or a first regionthereof) is selected from the group consisting of SEQ ID NOS: 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26 and 28, or is selected from the group consisting of at least10 contiguous monomers of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 and 28. Inother embodiments, the sequence of the oligomer (or of a first regionthereof) comprises one, two, or three base moieties that differ fromthose in oligomers having sequences of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26and 28,or the sequences of at least 10 contiguous monomers thereof, whenoptimally aligned with the selected sequence or region thereof.

In various embodiments, the sequence of the oligomer (or region thereof)is selected from the group consisting of SEQ ID NOs: 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,28, 129, 130, 131, 132, 133, 134, 135 and 136, or is selected from thegroup consisting of at least 10 contiguous monomers thereof. In otherembodiments, the sequence of the oligomer of the invention (or of afirst region thereof) comprises one, two, or three base moieties thatdiffer from those in oligomers having sequences of SEQ ID NOs: 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 28, 129, 130, 131, 132, 133, 134, 135 or 136, or the sequencesof at least 10 contiguous monomers thereof, when optimally aligned withthe selected sequence or region thereof.

In various embodiments, the sequence of the oligomer (or region thereof)is selected from the group consisting of SEQ ID NOs: 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159 and 160 or is selected from the groupconsisting of at least 10 contiguous monomers of SEQ ID NOs: 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159 and 160. In otherembodiments, the sequence of the oligomer (or of a first region thereof)comprises one, two, or three base moieties that differ from those inoligomers having sequences of SEQ ID NOs: 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159 or 160, or the sequences of at least 10contiguous monomers thereof when optimally aligned with the selectedsequence or region thereof.

In various embodiments the sequence of the oligomer (or region thereof)is selected from the group consisting of SEQ ID NOs: 111, 112, 114, 115,116, 118, 119, 122, 131, 132 and 136, or the sequence is selected fromthe group consisting of at least 10 contiguous monomers of SEQ ID NOs:111, 112, 114, 115, 116, 118, 119, 122, 131, 132 and 136, such as thesequence of 11, 12, 13, 14, 15 or 16 contiguous monomers thereof. Inother embodiments, the sequence of the oligomer (or of a region thereof)comprises one, two, or three base moieties that differ from those inoligomers having sequences of SEQ ID NOs: 111, 112, 114, 115, 116, 118,119, 122, 131, 132 or 136, or the sequences of at least 10 contiguousmonomers thereof, when optimally aligned with the selected sequence orregion thereof.

In various embodiments, the sequence of the oligomer (or region thereof)is selected from the group consisting of SEQ ID NOs: 149, 150, 153, 154,157, and 158, or the sequence is selected from the group consisting ofat least 10 contiguous monomers of SEQ ID NOs: 149, 150, 153, 154, 157,and 158, such as the sequence of 11, 12, 13, 14, 15 or 16 contiguousmonomers thereof. In other embodiments, the sequence of the oligomer ofthe invention (or of a region thereof) comprises one, two, or three basemoieties that differ from those in oligomers having sequences of SEQ IDNOs: 149, 150, 153, 154, 157, or 158, or the sequences of at least 10contiguous monomers thereof, when optimally aligned with the selectedsequence or region thereof.

In various embodiments, the sequence of the oligomer (or region thereof)is selected from the group consisting of SEQ ID NOs: 3, 4, 6, 7, 8, 10,11, 14, 23, 24 and 28, or the sequence is selected from the group of atleast 10 contiguous monomers of SEQ ID NOs: 3, 4, 6, 7, 8, 10, 11, 14,23, 24 and 28, such as the sequence of 11, 12, 13, 14, 15 or 16contiguous monomers thereof. In other embodiments, the sequence of theoligomer (or of a region thereof) comprises one, two, or three basemoieties that differ from those in oligomers having sequences of SEQ IDNOs: 3, 4, 6, 7, 8, 10, 11, 14, 23, 24 or 28, or the sequences of atleast 10 contiguous monomers thereof, when optimally aligned with theselected sequence or region thereof.

In various embodiments, the sequence of the oligomer (or region thereof)is selected from the group consisting of SEQ ID NOs: 151, 152, 155, 156,159 and 160; or the sequence is selected from the group of at least 10contiguous monomers of SEQ ID NOs: 151, 152, 155, 156, 159 and 160, suchas the sequence of 11, 12, 13, 14, 15 or 16 monomers thereof. In otherembodiments, the sequence of the oligomer (or of a region thereof)comprises one, two, or three base moieties that differ from those inoligomers having sequences of SEQ ID NOs: 151, 152, 155, 156, 159 and160, or the sequences of at least 10 contiguous monomers thereof, whenoptimally aligned with the selected sequence or region thereof.

In various embodiments, the sequence of the oligomer (or region thereof)is selected from the group consisting of SEQ ID NO: 57, 60, 64, 67, 70,74, 77, 82, 87, 90 and 96, or the sequence is selected from the group ofat least 10 contiguous monomers of SEQ ID NO: 57, 60, 64, 67, 70, 74,77, 82, 87, 90 and 96, such as the sequence of 11, 12, 13, 14, 15 or 16contiguous monomers thereof. In other embodiments, the sequence of theoligomer (or of a region thereof) comprises one, two, or three basemoieties that differ from those in oligomers having sequences of SEQ IDNO: 57, 60, 64, 67, 70, 74, 77, 82, 87, 90 and 96, or the sequences ofat least 10 contiguous monomers thereof, when optimally aligned with theselected sequence or region thereof.

In various embodiments, the sequence of the oligomer (or region thereof)is selected from the group consisting of SEQ ID NO: 87, 90 and 96, orthe sequence is selected from the group consisting of at least 10contiguous monomers of SEQ ID NO: 87, 90 and 96, such as the sequence of11, 12, 13, 14, 15 or 16 contiguous monomers thereof. In otherembodiments, the sequence of the oligomer (or of a region thereof)comprises one, two, or three base moieties that differ from those inoligomers having sequences of SEQ ID NO: 87, 90 and 96, or the sequencesof at least 10 contiguous monomers thereof, when optimally aligned withthe selected sequence or region thereof.

In various embodiments, the sequence of the oligomer (or region thereof)is selected from the group consisting of SEQ ID NO: 99, 100, 101, 102,103, and 104, or the sequence is selected from the group consisting ofat least 10 contiguous monomers of SEQ ID NO: 99, 100, 101, 102, 103,and 104, such as the sequence of 11, 12, 13, 14, 15 or 16 contiguousmonomers thereof. In various embodiments, the sequence of the oligomerof the invention (or of a first region thereof) comprises one, two, orthree base moieties that differ from those in oligomers having sequencesof SEQ ID NO: 99, 100, 101, 102, 103, and 104, or the sequences of atleast 10 contiguous monomers thereof, when optimally aligned with theselected sequence or region thereof.

In certain embodiments, the sequence of the oligomer (or region thereof)is selected from the group consisting of SEQ ID NO: 99, 100 and 104, orthe sequence is selected from the group consisting of at least 10contiguous monomers of SEQ ID NO: 99, 100 and 104. In variousembodiments, the sequence of the oligomer of the invention (or of afirst region thereof) comprises one, two, or three base moieties thatdiffer from those in oligomers having sequences of SEQ ID NO: 99, 100and 104, or the sequences of at least 10 contiguous monomers thereof,when optimally aligned with the selected sequence or region thereof.

In certain embodiments, the monomer region consists of 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 contiguousmonomers, such as 12-22, such as 12-18 monomers. Suitably, in someembodiments, the region is of the same length as the oligomer of theinvention.

In various embodiments, the oligomer comprises additional monomers atthe 5′ or 3′ ends, such as, independently, 1, 2, 3, 4 or 5 additionalmonomers at the 5′ and/or 3′ ends of the oligomer, which arenon-complementary to the target region. In various embodiments, theoligomer of the invention comprises a region which is complementary tothe target which is flanked 5′ and/or 3′ by additional monomers. In someembodiments, the additional monomers at the 5′ and/or 3′ ends are DNA orRNA monomers. In some embodiments, the additional monomers at the 5′and/or 3′ ends may represent region D as referred to in the context ofgapmer oligomers herein.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 2, such as SEQ ID NO 56, or according to a region of at least10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 3, such as SEQ ID NOs: 57, 58 or 59, or according to a regionof at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15or 16 contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 4, such as SEQ ID NOs: 60, 61 and 62, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In various embodiments the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 5, such as SEQ ID NO: 63, or according to a region of atleast 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 6, such as SEQ ID NO 64, 65 and 66, or according to a regionof at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15or 16 contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a sequence according to SEQID NO: 7, such as SEQ ID NOs: 67, 68 or 69, or according to a region ofat least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or16 contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 8, such as SEQ ID NOs: 70, 71 or 72, or according to a regionof at least 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15or 16 contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 9, such as SEQ ID NO: 73, or according to a region of atleast 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 10, such as SEQ ID NOs: 74, 75 or 76, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 11, such as SEQ ID NOs: 77, 78 or 79, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 12, such as SEQ ID NO: 80, or according to a region of atleast 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 13, such as SEQ ID NO: 81, or according to a region of atleast 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 14, such as SEQ ID NOs: 82, 83, or 84, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 15, such as SEQ ID NO: 85, or according to a region of atleast 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 16, such as SEQ ID NO: 86, or according to a region of atleast 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 17, such as SEQ ID NOs: 87, 88 or 89, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 18, such as SEQ ID NOs: 90, 91 or 92, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 19, such as SEQ ID NO: 93, or according to a region of atleast 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 20, such as SEQ ID NO: 94, or according to a region of atleast 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 21, such as SEQ ID NO: 95, or according to a region of atleast 10 contiguous monomers thereof, such as 11, 12, 13, 14, 15 or 16contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 22, such as SEQ ID NOs: 96, 97 or 98, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 23, such as SEQ ID NOs: 99, 137 or 138, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 24, such as SEQ ID NOs: 100, 139 or 140, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 25, such as SEQ ID NOs: 101, 141 or 142, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 26, such as SEQ ID NOs: 102, 143 or 144, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 27, such as SEQ ID NOs: 103, 145 or 146, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In various embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence according toSEQ ID NO: 28, such as SEQ ID NOs: 104, 147 or 148 or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a base sequence selected fromthe group consisting of SEQ ID NOs: 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159 and 160, as shown below in Table 1, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

TABLE 1 Oligomers Targeted to Variant PIK3CA Nucleic Acids SEQ ID NO:149* GCTCAGTGATTTXAGAGAGAGGAT SEQ ID NO: 150* TTTCTCCTGCTXAGTGATTTCAGASEQ ID NO: 151* GATTTXAGAGAGAGGA SEQ ID NO: 152* AGTGATTTXAGAGAGA SEQ IDNO: 153* ATCTTTCTCCTGCTXAGTGATTTC SEQ ID NO: 154*CAGTGATTTXAGAGAGAGGATCTC SEQ ID NO: 155* TCCTGCTXAGTGATTT SEQ ID NO:156* TTCTCCTGCTXAGTGA SEQ ID NO: 157† AGCCACCATGAXGTGCATCATTCA SEQ IDNO: 158† TCCAGCCACCATGAXGTGCATCAT SEQ ID NO: 159† GCCACCATGAXGTGCA SEQID NO: 160† ACCATGAXGTGCATCA *Where X is not C (i.e., X is A, G or T,preferably T). †Where X is not T (i.e., X is A, G or C, preferably C).

In some embodiments, the oligomer according to the invention consists ofor comprises monomers having a nucleobase sequence selected from thefollowing (5′-3′):

(G)(A)(T)TTXAG(A)(G)(A)(G) (SEQ ID NO: 161), wherein the monomers inparentheses are optionally present, and wherein X is not C (i.e., X isA, G or T, preferably T), for example, as shown in SEQ ID NOs: 149-152;

(C)(T)(G)CTXAG(T)(G)(G) (SEQ ID NO: 162), wherein the monomers inparentheses are optionally present, and wherein X is not C (i.e., X isA, G or T, preferably T), for example, as shown in SEQ ID NOs: 153-156;and

(C)(A)(T)GAXGT(G)(C)(A) (SEQ ID NO: 163), wherein the monomers inparentheses are optionally present, and wherein X is not T (i.e., X isA, G or C, preferably C), for example, as shown in SEQ ID NOs: 157-160.

In certain embodiments, X is in region B (of a gapmer or shortmer), andis, e.g., a DNA monomer. Suitably, X is positioned at the center ofregion B. In various embodiments, X is in region B and is flanked on the5′ end by at least 1, 2, 3 or 4 additional monomers of region B and/oris independently flanked on the 3′ end by at least 1, 2, 3 or 4additional monomers of region B. In some embodiments, X is not withinregions A, C or, where present, D. In other embodiments, X is withinregion B, but is not immediately adjacent to the monomers of regions Aor C.

In certain embodiments, the oligomer or region thereof, comprises one ormore base mismatches when compared to the sequence of the best-alignedtarget region of a nucleic acid which encodes the PIK3CA polypeptide.

In various embodiments, the oligomer of the invention does not comprisemore than four, such as not more than three, such as not more than two,such as not more than one base mismatch, when compared to the sequenceof the best-aligned target region of a nucleic acid which encodes thePIK3CA polypeptide, such as the target nucleic acid having the sequenceof SEQ ID NO: 1, or naturally occurring variants thereof. In certainembodiments, naturally occurring variants are allelic variants andspontaneous variants, such as nucleic acids which code for one or moreof the following amino acid mutations: E524K, E545K, or H1047R, such asnucleic acids which code for one of the following amino acid mutations:G1781A, G1790A or A3297G (the amino acid residue in front of theposition refers to the wild-type amino acid, the letter after theposition refers to the amino acid residue in a PIK3CA mutant).

In certain embodiments, the invention provides a method for thepreparation of an oligomer for the down-regulation of a target mRNAassociated with cancer cells, such as a PIK3CA mRNA target, the methodcomprising the steps of:

(a) identifying a single point mutation present in the target region ofa target mRNA associated with cancer, wherein the single point mutationis present in cancer cells but is absent in non-cancer cells; and

(b) preparing an oligomer which comprises a region of contiguousmonomers having a base sequence that is identical to the base sequenceof the reverse complement of the target region of the target mRNA.

Suitably, the single point mutation is associated with cancer, such asthe PIK3CA point mutations referred to herein.

Suitably, the oligomer is a gapmer or shortmer oligonucleotide asdescribed herein (although not limited necessarily to the oligomerstargeting PIK3CA). As referred to herein, in one embodiment, the monomerof the oligomer comprising the nucleobase that is complementary to thesingle point mutation in the mRNA target region is in region B.

In certain embodiments, the oligomers of the invention a target regionof the mRNA target nucleic acid which comprises a point mutationassociated with a cancer phenotype. In these embodiments, the oligomersof the invention suitably have a lower binding affinity to a targetregion of a target mRNA having a wild-type sequence, such as a PIK3CAmRNA found in non-cancerous cells, thereby selectively down-regulating(e.g., by inhibiting) the variant target nucleic acid in cancerous cellsand having less effect on the wild-type target nucleic acid innon-cancerous cells.

Nucleosides and Nucleoside Analogues

In various embodiments, at least one of the monomers present in theoligomer is a nucleoside analogue that contains a modified base, such asa base selected from 5-methylcytosine, isocytosine, pseudoisocytosine,5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine,diaminopurine, 2-chloro-6-aminopurine, xanthine and hypoxanthine.

In various embodiments, at least one of the monomers present in theoligomer is a nucleoside analogue that contains a modified sugar.

In some embodiments, the linkage between at least 2 contiguous monomersof the oligomer is other than a phosphodiester linkage.

In certain embodiments, the oligomer includes at least one monomer thathas a modified base, at least one monomer (which may be the samemonomer) that has a modified sugar and at least one inter-monomerlinkage that is non-naturally occurring.

Specific examples of nucleoside analogues are described by e.g. Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinionin Drug Development, 2000, 3(2), 293-213, and in Scheme 1 (in which somenucleoside analogues are shown as nucleotides):

The oligomer may thus comprise or consist of a simple sequence ofnatural occurring nucleosides—preferably 2DNA monomers, but alsopossibly RNA monomers, or a combination of nucleosides and one or morenucleoside analogues. In some embodiments, the nucleoside analoguesenhance the affinity of the oligomer for the target region of the targetnucleic acid.

Examples of suitable and preferred nucleoside analogues are provided byPCT/DK2006/000512, incorporated herein by reference in its entirety, orare referenced therein.

In some embodiments, the nucleoside analogue comprises a sugar moietymodified to provide a 2′-substituent group, such as 2′-O-alkyl-ribosesugars, 2′-amino-deoxyribose sugars, and 2′-fluoro-deoxyribose sugars.

In some embodiments, the nucleoside analogue comprises a sugar in whicha bridged structure, creating a bicyclic sugar (LNA), which enhancesbinding affinity and may also provide some increased nucleaseresistance. In various embodiments, the LNA monomer is selected fromoxy-LNA (such as beta-D-oxy-LNA, and alpha-L-oxy-LNA), and/or amino-LNA(such as beta-D-amino-LNA and alpha-L-amino-LNA) and/or thio-LNA (suchas beta-D-thio-LNA and alpha-L-thio-LNA) and/or ENA (such as beta-D-ENAand alpha-L-ENA). In certain embodiments, the LNA monomers arebeta-D-oxy-LNA. LNA monomers are further described below.

In various embodiments, incorporation of affinity-enhancing nucleosideanalogues in the oligomer, such as LNA monomers or monomers containing2′-substituted sugars, or incorporation of modified linkage groupsprovides increased nuclease resistance. In various embodiments,incorporation of affinity-enhancing nucleoside analogues allows the sizeof the oligomer to be reduced, and also reduces the size of the oligomerthat binds specifically to a target region of a target sequence.

In some embodiments, the oligomer comprises at least 2 nucleosideanalogues. In some embodiments, the oligomer comprises from 3-8nucleoside analogues, e.g. 6 or 7 nucleoside analogues. In variousembodiments, at least one of the nucleoside analogues is a lockednucleic acid (LNA) monomer; for example at least 3 or at least 4, or atleast 5, or at least 6, or at least 7, or 8, nucleoside analogues areLNA monomers. In some embodiments, all the nucleoside analogues are LNAmonomers.

It will be recognised that when referring to a preferred oligomer basesequence, in certain embodiments, the oligomers comprise a correspondingnucleoside analogue, such as a corresponding LNA monomer or othercorresponding nucleoside analogue, which raise the duplex stability(T_(m)) of the oligomer/target region duplex (i.e. affinity enhancingnucleoside analogues).

In various embodiments, any mismatches (i.e., non-complementarities)between the base sequence of the oligomer and the base sequence of thetarget region, if present, are preferably located other than in theregions of the oligomer that contain affinity-enhancing nucleosideanalogues (e.g., regions A or C), such as within region B as referred toherein, and/or within region D as referred to herein, and/or in regionsconsisting of DNA monomers, and/or in regions which are 5′ or 3′ to theregion of the oligomer that is complementary to the target region.

In some embodiments the nucleoside analogues present within the oligomerof the invention (such as in regions A and C mentioned herein) areindependently selected from, for example: monomers containing2′-O-alkyl-ribose sugars, monomers containing 2′-amino-deoxyribosesugars, monomers containing 2′-fluoro-deoxyribose sugars, LNA monomers,monomers containing arabinose sugars (“ANA monomers”), monomerscontaining 2′-fluoro-arabinose sugars, monomers containingd-arabino-hexitol sugars (“HNA monomers”), intercalating monomers asdefined in Christensen (2002) Nucl. Acids. Res. 30: 4918-4925, herebyincorporated by reference, and 2′-O-methoxyethyl-ribose (2′MOE) sugars.In some embodiments, there is only one of the above types of nucleosideanalogues present in the oligomer of the invention, or region thereof.

In certain embodiments, the nucleoside analogues contain 2′MOE sugars,2′-fluoro-deoxyribose sugars, or LNA sugars, and as such theoligonucleotide of the invention may comprise nucleoside analogues whichare independently selected from these three types. In certain oligomerembodiments containing nucleoside analogues, at least one of saidnucleoside analogues contains a 2′-MOE-ribose sugar, such as 2, 3, 4, 5,6, 7, 8, 9 or 10 nucleoside analogues containing 2′-MOE-ribose sugars.In some embodiments, at least one nucleoside analogue contains a2′-fluoro-deoxyribose sugar, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10nucleoside analogues containing 2′-fluoro-DNA nucleoside sugars.

In various embodiments, the oligomer according to the inventioncomprises at least one Locked Nucleic Acid (LNA) monomer, such as 1, 2,3, 4, 5, 6, 7, or 8 LNA monomers, such as 3-7 or 4 to 8 LNA monomers, or3, 4, 5, 6 or 7 LNA monomers. In various embodiments, all the nucleosideanalogues are LNA monomers. In certain embodiments, the oligomercomprises both beta-D-oxy-LNA monomers, and one or more of the followingLNA monomers: thio-LNA monomers, amino-LNA monomers, oxy-LNA monomers,and/or ENA monomers in either the beta-D or alpha-L configurations, orcombinations thereof. In certain embodiments, the cytosine base moietiesof all LNA monomers in the oligomer are 5-methylcytosines. In certainembodiments of the invention, the oligomer comprises both LNA and DNAmonomers. Typically, the combined total of LNA and DNA monomers is10-25, preferably 10-20, even more preferably 12-16. In some embodimentsof the invention, the oligomer or region thereof consists of at leastone LNA monomer, and the remaining monomers are DNA monomers. In certainembodiments, the oligomer comprises only LNA monomers and nucleosides(such as RNA or DNA monomers, most preferably DNA monomers) optionallywith modified linkage groups such as phosphorothioate.

In various embodiments, at least one of the nucleoside analogues presentin the oligomer has a modified base selected from the group consistingof 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine,and 2-chloro-6-aminopurine.

LNA

The term “LNA monomer” refers to a nucleoside analogue containing abicyclic sugar (an “LNA sugar”). The terms “LNA oligonucleotide” and“LNA oligomer” refer to an oligomer containing one or more LNA monomers.

The LNA used in the oligonucleotide compounds of the inventionpreferably has the structure of the general formula I

wherein X is selected from —O—, —S—, —N(R^(N)*)—, —C(R⁶R⁶*)—;

B is selected from hydrogen, optionally substituted C₁₋₄-alkoxy,optionally substituted C₁₋₄-alkyl, optionally substituted C₁₋₄-acyloxy,nucleobases, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups, andligands;

P designates the radical position for an internucleoside linkage to asucceeding monomer, or a 5′-terminal group, such internucleoside linkageor 5′-terminal group optionally including the substituent R⁵ or equallyapplicable the substituent R⁵*;

P* designates an internucleoside linkage to a preceding monomer, or a3′-terminal group;

R⁴* and R²* together designate a biradical consisting of 1-4groups/atoms selected from —C(R^(a)R^(b))—, —C(R^(a))═C(R^(b))—,—C(R^(a))═N—, —O—, —Si(R^(a))₂—, —S—, —SO₂—, —N(R^(a))—, and >C=Z,

wherein Z is selected from —O—, —S—, and —N(R^(a))—, and R^(a) and R^(b)each is independently selected from hydrogen, optionally substitutedC₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl, optionallysubstituted C₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl,C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl,formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl- aminocarbonyl, mono-and di(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl,C₁₋₆-alkyl-carbonylamino, carbamido, C₁₋₆-alkanoyloxy, sulphono,C₁₋₆-alkylsulphonyloxy, nitro, azido, sulphanyl, C₁₋₆-alkylthio,halogen, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups, andligands, where aryl and heteroaryl are optionally substituted and wheretwo geminal substituents R^(a) and R^(b) together may designateoptionally substituted methylene (═CH₂), and each of the substituentsR¹*, R², R³, R⁵, R⁵*, R⁶ and R⁶*, which are present is independentlyselected from hydrogen, optionally substituted C₁₋₁₂-alkyl, optionallysubstituted C₂₋₁₂-alkenyl, optionally substituted C₂₋₁₂-alkynyl,hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl, C₂₋₁₂-alkenyloxy, carboxy,C₁₋₁₂-alkoxycarbonyl, C₂₋₁₂-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,hetero-aryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted, and where two geminal substituents together maydesignate oxo, thioxo, imino, or optionally substituted methylene, ortogether may form a spiro biradical consisting of a 1-5 carbon atom(s)alkylene chain which is optionally interrupted and/or terminated by oneor more heteroatoms/groups selected from —O—, —S—, and —(NR^(N))— whereR^(N) is selected from hydrogen and C₁₋₄-alkyl, and where two adjacent(non-geminal) substituents may designate an additional bond resulting ina double bond; and R^(N)*, when present and not involved in a biradical,is selected from hydrogen and C₁₋₄-alkyl; and basic salts and acidaddition salts thereof;

In some embodiments R⁵* is selected from H, —CH₃, —CH₂—CH₃, —CH₂—O—CH₃,and —CH═CH₂.

In various embodiments, R⁴* and R²* together designate a biradicalselected from —C(R^(a)R^(b))—O—, —C(R^(a)R^(b))—C(R^(c)R^(d))—O—,—C(R^(a)R^(b))—C(R^(c)R^(d))—C(R^(e)R^(f))—O—,—C(R^(a)R^(b))—O—C(R^(c)R^(d))—, —C(R^(a)R^(b))—O—C(R^(c)R^(d))—O—,—C(R^(a)R^(b))—C(R^(c)R^(d))—,—C(R^(a)R^(b))—C(R^(c)R^(d))—C(R^(e)R^(f))—,—C(R^(a))═C(R^(b))—C(R^(c)R^(d))—, —C(R^(a)R^(b))—N(R^(c))—,—C(R^(a)R^(b))—C(R^(c)R^(d))—N(R^(e))—, —C(R^(a)R^(b))—N(R^(c))—O—, and—C(R^(a)R^(b))—S—, —C(R^(a)R^(b))—C(R^(c)R^(d))—S—, wherein R^(a),R^(b), R^(c), R^(d), R^(e), and R^(f) each is independently selectedfrom hydrogen, optionally substituted C₁₋₁₂-alkyl, optionallysubstituted C₂₋₁₂-alkenyl, optionally substituted C₁₋₁₂-alkynyl,hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl, C₂₋₁₂-alkenyloxy, carboxy,C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted and where two geminal substituents R^(a) andR^(b) together may designate optionally substituted methylene (═CH₂),

In a further embodiment R⁴* and R²* together designate a biradical(bivalent group) selected from —CH₂—O—, —CH₂—S—, —CH₂—NH—, —CH₂—N(CH₃)—,—CH₂—CH₂—O—, —CH₂—CH(CH₃)—, —CH₂—CH₂—S—, —CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—,—CH₂-CH₂—CH₂—O—, —CH₂—CH₂—CH(CH₃)—, —CH═CH—CH₂—, —CH₂—O—CH₂—O—,—CH₂—NH—O—, —CH₂—N(CH₃)—O—, —CH₂—O—CH₂—, —CH(CH₃)—O—, —CH(CH₂—O—CH₃)—O—.

For all chiral centers, asymmetric groups may be found in either R or Sorientation.

Preferably, the LNA monomer used in the oligomer of the inventioncomprises at least one LNA monomer according to any of the formulas

wherein Y is —O—, —O—CH₂—, —S—, —NH—, or N(R^(H)); Z and Z* areindependently selected among an internucleoside linkage, a terminalgroup or a protecting group; B constitutes a natural or non-natural basemoiety, and R^(H) is selected from hydrogen and C₁₋₄-alkyl.

Specifically preferred LNA monomers are shown in Scheme 2:

The term “thio-LNA” refers to an LNA monomer in which Y in the generalformula above is selected from S or —CH₂—S—. Thio-LNA can be in eitherthe beta-D or the alpha-L-configuration.

The term “amino-LNA” refers to an LNA monomer in which Y in the generalformula above is selected from —N(H)—, N(R)—, CH₂—N(H)—, and —CH₂—NR)—where R is selected from hydrogen and C₁₋₄-alkyl. Amino-LNA can be ineither the beta-D or the alpha-L-configuration.

The term “oxy-LNA” refers to an LNA monomer in which Y in the generalformula above represents —O— or —CH₂—O—. Oxy-LNA can be in either thebeta-D or the alpha-L-configuration.

The term “ENA” refers to an LNA monomer in which Y in the generalformula above is —CH₂—O— (where the oxygen atom of —CH₂—O— is attachedto the 2′-position relative to the base B).

In a preferred embodiment the LNA monomer is selected from abeta-D-oxy-LNA monomer, an alpha-L-oxy-LNA monomer, a beta-D-amino-LNAmonomer and beta-D-thio-LNA monomer, in particular a beta-D-oxy-LNAmonomer.

In the present context, the term C₁₋₄-alkyl means a linear or branchedsaturated hydrocarbon chain wherein the chain has from one to fourcarbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl and tert-butyl.

RNAse H Recruitment

In some embodiments, the oligomer functions via non-RNase-mediateddegradation of a target mRNA, such as by steric hindrance oftranslation, or other mechanisms; however, in various embodiments, theoligomers of the invention are capable of recruiting an endoribonuclease(RNase), such as RNase H.

Typically, the oligomer comprises of a region of at least 6, such as atleast 7 contiguous monomers, such as at least 8 or at least 9 contiguousmonomers, including 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguousmonomers, which, when forming a duplex with the target region of thetarget RNA, is capable of recruiting RNase. The region of the oligomerwhich is capable of recruiting RNAse may be region B, as referred to inthe context of a gapmer as described herein. In some embodiments, theregion of the oligomer which is capable of recruiting RNAse, such asregion B, consists of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20monomers.

EP 1 222 309 provides in vitro methods for determining RNaseH activity,which may be used to determine the ability of the oligomers of theinvention to recruit RNaseH. An oligomer is deemed capable of recruitingRNase H if, when contacted with the complementary region of the RNAtarget, it has an initial rate, as measured in pmol/1/min, of at least1%, such as at least 5%, such as at least 10% or less than 20% of anoligonucleotide having the same base sequence but containing only DNAmonomers, with no 2′ substitutions, with phosphorothioate linkage groupsbetween all monomers in the oligonucleotide, using the methodologyprovided in Examples 91-95 of EP 1 222 309, incorporated herein byreference.

In some embodiments, an oligomer is deemed essentially incapable ofrecruiting RNaseH if, when contacted with the target region of the RNAtarget, and RNaseH, the RNaseH initial rate, as measured in pmol/1/min,is less than 1%, such as less than 5%,such as less than 10% or less than20% of the initial rate determined using an oligonucleotide having thesame base sequence, but containing only DNA monomers, with no 2′substitutions, with phosphorothioate linkage groups between all monomersin the oligonucleotide, using the methodology provided in Examples 91-95of EP 1 222 309.

In other embodiments, an oligomer is deemed capable of recruiting RNaseHif, when contacted with the target region of the RNA target, and RNaseH,the RNaseH initial rate, as measured in pmol/1/min, is at least 20%,such as at least 40%, such as at least 60%, such as at least 80% of theinitial rate determined using an oligonucleotide having the same basesequence, but containing only DNA monomers, with no 2′ substitutions,with phosphorothioate linkage groups between all monomers in theoligonucleotide, using the methodology provided in Examples 91-95 of EP1 222 309.

Typically, the region of the oligomer which forms the duplex with thecomplementary target region of the target RNA and is capable ofrecruiting RNase contains DNA monomers and LNA monomers and forms aDNA/RNA-like duplex with the target region. The LNA monomers arepreferably in the alpha-L configuration, particularly preferred beingalpha-L-oxy LNA.

In various embodiments, the oligomer of the invention comprises bothnucleosides and nucleoside analogues, and may be in the form of agapmer, a headmer or a mixmer.

A “headmer” is defined as an oligomer that comprises a first region anda second region that is contiguous thereto, with the 5′-most monomer ofthe second region linked to the 3′-most monomer of the first region. Thefirst region comprises a contiguous stretch of non-RNase-recruitingnucleoside analogues, and the second region comprises a contiguousstretch (such as at least 7 contiguous monomers) of DNA monomers ornucleoside analogue monomers recognizable and cleavable by the RNAse.

A “tailmer” is defined as an oligomer that comprises a first region anda second region that is contiguous thereto, with the 5′-most monomer ofthe second region linked to the 3′-most monomer of the first region. Thefirst region comprises a contiguous stretch (such as at least 7 suchmonomers) of DNA monomers or nucleoside analogue monomers recognizableand cleavable by the RNase, and the second region comprises a contiguousstretch of non-RNase recruiting nucleoside analogue monomers.

Other “chimeric” oligomers, called “mixmers”, consist of an alternatingcomposition of (i) DNA monomers or nucleoside analogue monomersrecognizable and cleavable by RNase, and (ii) non-RNase recruitingnucleoside analogue monomers.

In some embodiments, in addition to enhancing affinity of the oligomerfor the target region, some nucleoside analogues also mediate RNase(e.g., RNase H) binding and cleavage. Since •-L-LNA monomers recruitRNase activity to a certain extent, in some embodiments, gap regions(e.g., region B as referred to herein below) of oligomers containing•-L-LNA monomers consist of fewer monomers recognizable and cleavable bythe RNase, and more flexibility in the mixmer construction isintroduced.

Conjugates

In the context of this disclosure, the term “conjugate” indicates acompound formed by the covalent attachment (“conjugation”) of anoligomer as described herein, to one or more moieties that are notthemselves nucleic acids or monomers (“conjugated moieties”). Examplesof such conjugated moieties include macromolecular compounds such asproteins, fatty acid chains, sugar residues, glycoproteins, polymers, orcombinations thereof. Typically proteins may be antibodies for a targetprotein. Typical polymers may be polyethylene glycol.

Accordingly, provided herein are conjugates comprising an oligomer asherein described, and at least one conjugated moiety that is not anucleic acid or monomer, covalently attached to said oligomer.Therefore, in certain embodiments where the oligomer of the inventionconsists of contiguous monomers having a specified sequence of bases, asherein disclosed, the conjugate may also comprise at least oneconjugated moiety that is covalently attached to the oligomer.

In various embodiments of the invention, the oligomer is conjugated to amoiety that increases the cellular uptake of oligomeric compounds.WO2007/031091 provides suitable ligands and conjugates, which are herebyincorporated by reference.

In various embodiments, conjugation (to a conjugated moiety) may enhancethe activity, cellular distribution or cellular uptake of the oligomerof the invention. Such moieties include, but are not limited to,antibodies, polypeptides, lipid moieties such as a cholesterol moiety,cholic acid, a thioether, e.g. Hexyl-s-tritylthiol, a thiocholesterol,an aliphatic chain, e.g., dodecandiol or undecyl residues, aphospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-o-hexadecyl-rac-glycero-3-h-phosphonate, a polyamine or apolyethylene glycol chain, an adamantane acetic acid, a palmityl moiety,an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

In certain embodiments, the oligomers of the invention are conjugated toactive drug substances, for example, aspirin, ibuprofen, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic.

In certain embodiments the conjugated moiety is a sterol, such ascholesterol.

In various embodiments, the conjugated moiety comprises or consists of apositively charged polymer, such as a positively charged peptides of,for example 1-50, such as 2-20 such as 3-10 amino acid residues inlength, and/or polyalkylene oxide such as polyethylene glycol (PEG) orpolypropylene glycol—see WO 2008/034123, hereby incorporated byreference. Suitably the positively charged polymer, such as apolyalkylene oxide may be attached to the oligomer of the invention viaa linker such as the releasable linker described in WO 2008/034123.

By way of example, the following moieties may be used in the conjugatesof the invention:

Activated Oligomers

The term “activated oligomer,” as used herein, refers to an oligomer ofthe invention that is covalently linked (i.e., functionalized) to atleast one functional moiety that permits covalent linkage of theoligomer to one or more conjugated moieties, i.e., moieties that are notthemselves nucleic acids or monomers, to form the conjugates hereindescribed. Typically, a functional moiety will comprise a chemical groupthat is capable of covalently bonding to the oligomer via, e.g., a3′-hydroxyl group or the exocyclic NH₂ group of the adenine base, aspacer that is preferably hydrophilic and a terminal group that iscapable of binding to a conjugated moiety (e.g., an amino, sulfhydryl orhydroxyl group). In some embodiments, this terminal group is notprotected, e.g., is an NH₂ group. In other embodiments, the terminalgroup is protected, for example, by any suitable protecting group suchas those described in “Protective Groups in Organic Synthesis” byTheodora W. Greene and Peter G. M. Wuts, 3rd edition (John Wiley & Sons,1999). Examples of suitable hydroxyl protecting groups include esterssuch as acetate ester, aralkyl groups such as benzyl, diphenylmethyl, ortriphenylmethyl, and tetrahydropyranyl. Examples of suitable aminoprotecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl,triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acyl groupssuch as trichloroacetyl or trifluoroacetyl.

In some embodiments, the functional moiety is self-cleaving. In otherembodiments, the functional moiety is biodegradable. See e.g., U.S. Pat.No. 7,087,229, which is incorporated by reference herein in itsentirety.

In some embodiments, oligomers of the invention are functionalized atthe 5′ end in order to allow covalent attachment of the conjugatedmoiety to the 5′ end of the oligomer. In other embodiments, oligomers ofthe invention can be functionalized at the 3′ end. In still otherembodiments, oligomers of the invention can be functionalized along thebackbone or on the heterocyclic base moiety. In yet other embodiments,oligomers of the invention can be functionalized at more than oneposition independently selected from the 5′ end, the 3′ end, thebackbone and the base.

In some embodiments, activated oligomers of the invention aresynthesized by incorporating during the synthesis one or more monomersthat is covalently attached to a functional moiety. In otherembodiments, activated oligomers of the invention are synthesized withmonomers that have not been functionalized, and the oligomer isfunctionalized upon completion of synthesis.

In some embodiments, the oligomers are functionalized with a hinderedester containing an aminoalkyl linker, wherein the alkyl portion has theformula (CH₂)_(w), wherein w is an integer ranging from 1 to 10,preferably about 6, wherein the alkyl portion of the alkylamino groupcan be straight chain or branched chain, and wherein the functionalgroup is attached to the oligomer via an ester group(—O—C(O)—(CH₂)_(w)NH).

In other embodiments, the oligomers are functionalized with a hinderedester containing a (CH₂)_(w)-sulfhydryl (SH) linker, wherein w is aninteger ranging from 1 to 10, preferably about 6, wherein the alkylportion of the alkylamino group can be straight chain or branched chain,and wherein the functional group attached to the oligomer via an estergroup (—O—C(O)—(CH₂)_(w)SH) In some embodiments, sulfhydryl-activatedoligonucleotides are conjugated with polymer moieties such aspolyethylene glycol or peptides (via formation of a disulfide bond).

Activated oligomers containing hindered esters as described above can besynthesized by any method known in the art, and in particular, bymethods disclosed in PCT Publication No. WO 2008/034122 and the examplestherein, which is incorporated herein by reference in its entirety.

Activated oligomers covalently linked to at least one functional moietycan be synthesized by any method known in the art, and in particular, bymethods disclosed in U.S. Patent Publication No. 2004/0235773, which isincorporated herein by reference in its entirety, and in Zhao et al.(2007) J. Controlled Release 119:143-152; and Zhao et al. (2005)Bioconjugate Chem. 16:758-766.

In still other embodiments, the oligomers of the invention arefunctionalized by introducing sulfhydryl, amino or hydroxyl groups intothe oligomer by means of a functionalizing reagent substantially asdescribed in U.S. Pat. Nos. 4,962,029 and 4,914,210, i.e., asubstantially linear reagent having a phosphoramidite at one end linkedthrough a hydrophilic spacer chain to the opposing end which comprises aprotected or unprotected sulfhydryl, amino or hydroxyl group. Suchreagents primarily react with hydroxyl groups of the oligomer. In someembodiments, such activated oligomers have a functionalizing reagentcoupled to a 5′-hydroxyl group of the oligomer. In other embodiments,the activated oligomers have a functionalizing reagent coupled to a3′-hydroxyl group. In still other embodiments, the activated oligomersof the invention have a functionalizing reagent coupled to a hydroxylgroup on the backbone of the oligomer. In yet further embodiments, theoligomer of the invention is functionalized with more than one of thefunctionalizing reagents as described in U.S. Pat. Nos. 4,962,029 and4,914,210, incorporated herein by reference in their entirety. Methodsof synthesizing such functionalizing reagents and incorporating theminto monomers or oligomers are disclosed in U.S. Pat. Nos. 4,962,029 and4,914,210.

In some embodiments, the 5′-terminus of a solid-phase bound oligomer isfunctionalized with a dienyl phosphoramidite derivative, followed byconjugation of the deprotected oligomer with, e.g., an amino acid orpeptide via a Diels-Alder cycloaddition reaction.

In various embodiments, the incorporation of monomers containing2′-sugar modifications, such as a 2′-carbamate substituted sugar or a2′-(O-pentyl-N-phthalimido)-deoxyribose sugar into the oligomerfacilitates covalent attachment of conjugated moieties to the sugars ofthe oligomer. In other embodiments, an oligomer with an amino-containinglinker at the 2′-position of one or more monomers is prepared using areagent such as, for example,5′-dimethoxytrityl-2′-O-(e-phthalimidylaminopentyl)-2′-deoxyadenosine-3′-N,N-diisopropyl-cyanoethoxyphosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters,1991, 34, 7171.

In still further embodiments, the oligomers of the invention haveamine-containing functional moieties on the nucleobase, including on theN6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or5 positions of cytosine. In various embodiments, such functionalizationmay be achieved by using a commercial reagent that is alreadyfunctionalized in the oligomer synthesis.

Some functional moieties are commercially available, for example,heterobifunctional and homobifunctional linking moieties are availablefrom the Pierce Co. (Rockford, Ill.). Other commercially availablelinking groups are 5′-Amino-Modifier C6 and 3′-Amino-Modifier reagents,both available from Glen Research Corporation (Sterling, Va.).5′-Amino-Modifier C6 is also available from ABI (Applied BiosystemsInc., Foster City, Calif.) as Aminolink-2, and 3′-Amino-Modifier is alsoavailable from Clontech Laboratories Inc. (Palo Alto, Calif.).

Compositions

In various embodiments, the oligomer of the invention, or a conjugatethereof, is used in pharmaceutical formulations and compositions.Suitably, such compositions comprise a pharmaceutically acceptablediluent, carrier, salt or adjuvant.

Suitable dosages, formulations, administration routes, compositions,dosage forms, combinations with other therapeutic agents, pro-drugformulations are also provided in PCT/DK2006/000512—which are herebyincorporated by reference. Details on techniques for formulation andadministration also may be found in the latest edition of “REMINGTON'SPHARMACEUTICAL SCIENCES” (Maack Publishing Co, Easton Pa.).

In some embodiments, an oligomer of the invention is covalently linkedto a conjugated moiety to aid in delivery of the oligomer across cellmembranes. An example of a conjugated moiety that aids in delivery ofthe oligomer across cell membranes is a lipophilic moiety, such ascholesterol. In various embodiments, an oligomer of the invention isformulated with lipid formulations that form liposomes, such asLipofectamine 2000 or Lipofectamine RNAiMAX, both of which arecommercially available from Invitrogen. In some embodiments, theoligomers of the invention are formulated with a mixture of one or morelipid-like non-naturally occurring small molecules (“lipidoids”).Libraries of lipidoids can be synthesized by conventional syntheticchemistry methods and various amounts and combinations of lipidoids canbe assayed in order to develop a vehicle for effective delivery of anoligomer of a particular size to the targeted tissue by the chosen routeof administration. Suitable lipidoid libraries and compositions can befound, for example in Akinc et al, Nature Biotechnol., 26, 561-569(2008), which is incorporated by reference herein.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts that retain the desired biological activity of the hereinidentified compounds and exhibit acceptable levels of undesired toxiceffects. Non-limiting examples of such salts can be formed with organicamino acid and base addition salts formed with metal cations such aszinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt,nickel, cadmium, sodium, potassium, and the like, or with a cationformed from ammonia, N,N′-dibenzylethylene-diamine, D-glucosamine,tetraethylammonium, or ethylenediamine; or (c) combinations of (a) and(b); e.g., a zinc tannate salt or the like.

Applications

The term “treatment” as used herein refers to both treatment of anexisting disease (e.g., a disease or disorder as referred to hereinbelow), or prevention of a disease, i.e., prophylaxis. It will thereforebe recognised that, in certain embodiments, “treatment” includesprophylaxis.

In various embodiments, the oligomers of the invention may be utilizedas research reagents for, for example, diagnostics, therapeutics andprophylaxis.

In some embodiments, such oligomers may be used for research purposes tospecifically inhibit the expression of PIK3CA protein (typically, bydegrading or inhibiting the PIK3CA mRNA and thereby preventing proteinformation) in cells and experimental animals, thereby facilitatingfunctional analysis of the target or an appraisal of its usefulness as atarget for therapeutic intervention.

In certain embodiments, the oligomers may be used in diagnostics todetect and quantitate PIK3CA expression in cells and tissues by northernblotting, in-situ hybridisation or similar techniques.

In various therapeutic embodiments, a non-human animal or a human,suspected of having a disease or disorder, which can be treated bymodulating the expression of PIK3CA is treated by administering aneffective amount of an oligomer (or conjugate thereof) in accordancewith this invention. Further provided are methods of treating a mammal,such as treating a human, suspected of having or being prone to adisease or condition, associated with expression of PIK3CA byadministering a therapeutically or prophylactically effective amount ofone or more of the oligomers or compositions of the invention.

In various therapeutic embodiments, a non-human animal or a humansuspected of having a disease or disorder which can be treating bymodulating the expression of PIK3CA and of beta-catenin is treated byadministering an effective amount of an oligomer (or conjugate thereof)in accordance with this invention.

In various therapeutic embodiments, the non-human animal or human istreated with more than one oligomer of the invention (or conjugate),wherein one oligomer preferably binds to a PIK3CA target region and asecond oligomer preferably binds to a beta-catenin target region.

In certain embodiments, the invention also provides for the use of thecompounds or conjugates of the invention as described for themanufacture of a medicament for the treatment of a disorder as referredto herein, or for a method of the treatment of a disorder as referred toherein.

In various embodiments, the invention also provides for a method fortreating a disorder as referred to herein said method comprisingadministering a compound according to the invention as herein described,and/or a conjugate according to the invention, and/or a pharmaceuticalcomposition according to the invention to a patient in need thereof.

In various embodiments, the invention relates to an oligomer, acomposition or a conjugate thereof as described herein for use as amedicament.

In various embodiments, the invention provides for a method for treatinga disorder as referred to herein, the method comprising administering aneffective amount of a compound according to the invention as hereindescribed, and/or an effective amount of a conjugate according to theinvention, and/or a pharmaceutical composition according to theinvention to a patient in need thereof.

In various embodiments, the oligomer, or conjugate thereof, induces adesired therapeutic effect in humans through, for example, hydrogenbonding to a target nucleic acid. The oligomer causes a decrease (e.g.,inhibition) in the expression of a target via hydrogen bonding (e.g.,hybridisation) to the mRNA of the target thereby resulting in areduction in gene expression.

It is highly preferred that the compounds of the invention are capableof hybridising to the target nucleic acid, such as PIK3CA mRNA, byWatson-Crick base pairing.

Medical Indications

In some embodiments, disorder to be treated is a hyperproliferativedisorder, such as cancer, which is a solid tumor. In variousembodiments, the cancer is a carcinoma. In certain embodiments, thecancer is a sarcoma. In yet further embodiments, the cancer is a glioma.

In certain embodiments, the carcinoma is selected from the groupconsisting of malignant melanoma, basal cell carcinoma, ovariancarcinoma, breast carcinoma, non-small cell lung cancer, renal cellcarcinoma, bladder carcinoma, recurrent superficial bladder cancer,stomach carcinoma, prostatic carcinoma, pancreatic carcinoma, lungcarcinoma, cervical carcinoma, cervical dysplasia, laryngealpapillomatosis, colon carcinoma, colorectal carcinoma and carcinoidtumors.

In various embodiments, the carcinoma is selected from the groupconsisting of malignant melanoma, non-small cell lung cancer, breastcarcinoma, colon carcinoma and renal cell carcinoma. In still furtherembodiments, the carcinoma is a malignant melanoma selected from thegroup consisting of superficial spreading melanoma, nodular melanoma,lentigo malignant melanoma, acral melanoma, amelanotic melanoma anddesmoplastic melanoma.

In various embodiments, the sarcoma is selected from the groupconsisting of osteosarcoma, Ewing's sarcoma, chondrosarcoma, malignantfibrous histiocytoma, fibrosarcoma and Kaposi's sarcoma.

In some embodiments the solid tumor is selected from the groupconsisting of glioblastoma, malignant melanoma, medulloblastoma,hepatocellular carcinoma, head and neck squamous cell carcinoma,gastric, ovarian, cervix and colorectal cancers, cancers of the breast,lung and colon, large B-cell lymphoma, anaplastic astrocytoma,anaplastic oligodendroglioma, prostate cancer, endometrial cancer,pancreatic cancer, bowel cancer, leukaemia, esophagus cancer, andthyroid cancer. In some embodiments, the solid tumor is liver or kidneycancer.

In various embodiments, the cancer is selected from the group consistingof colorectal, glioblastoma, gastric, hepatocellular, breast, ovarianand lung cancer.

In various embodiments, the cancer is selected from the group consistingof; non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia (e.g., acuteleukemia such as acute lymphocytic leukemia, acute myelocytic leukemia,chronic myeloid leukemia, chronic lymphocytic leukemia, multiplemyeloma), colon carcinoma, rectal carcinoma, pancreatic cancer, breastcancer, ovarian cancer, prostate cancer, renal cell carcinoma, hepatoma,bile duct carcinoma, choriocarcinoma, cervical cancer, testicularcancer, lung carcinoma, bladder carcinoma, melanoma, head and neckcancer, brain cancer, cancers of unknown primary site, neoplasms,cancers of the peripheral nervous system, cancers of the central nervoussystem, tumors (e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, small cell lung carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma), heavychain disease, metastases, and any disease or disorder characterized byuncontrolled or abnormal cell growth.

In some embodiments, the cancer is selected from the group consisting ofHodgkin's lymphoma, leukaemia, such as acute lymphocytic leukaemia,colon carcinoma, rectal carcinoma, brain cancer, neural blastomas, lungcancer, pancreatic cancer, melanoma, acute myelogenous leukaemia, livercancer, thyroid cancer, kidney cancer, urinary tract cancer and bladdercancer.

In some embodiments, the cancer is selected from the group consisting ofHodgkin's lymphoma, leukaemia, such as acute lymphocytic leukaemia,colon carcinoma, brain cancer, and neural blastomas.

In various embodiments, the cancer is selected from the group consistingof lung, breast, colon, prostate, pancreas, lung, liver, thyroid,kidney, brain, testes, stomach, intestine, bowel, spinal cord, sinuses,bladder, urinary tract and ovarian cancer.

In certain embodiments, for example for the treatment of brain cancer,the oligomer or conjugate of the invention does not comprisephosphorothioate linkages between adjacent monomers.

In certain embodiments, the disease or disorder is associated with amutation in the PIK3CA gene or a gene whose protein product isassociated with or interacts with PIK3CA. Therefore, in variousembodiments, the target mRNA is a mutated form of the PIK3CA sequence;for example, it comprises one or more single point mutations, such asSNPs associated with cancer.

In certain embodiments, the disease or disorder is associated withabnormal levels of a mutated form of PIK3CA. In certain embodiments, thedisease or disorder is associated with abnormal levels of a wild-typeform of PIK3CA. One aspect of the invention is directed to a method oftreating a mammal suffering from or susceptible to conditions associatedwith abnormal levels of PIK3CA, comprising administering to the mammal atherapeutically effective amount of an oligomer of the inventiontargeted to PIK3CA or various conjugates thereof. In some embodiments,the oligomer comprises one or more LNA units. Another aspect of theinvention is directed to a method of treating a mammal suffering from orsusceptible to conditions associated with abnormal levels of PIK3CA,comprising administering to the mammal a therapeutically effectiveamount of an oligomer of the invention targeted to PIK3CA and tobeta-catenin or various conjugates thereof.

Suitable dosages, formulations, administration routes, compositions,dosage forms, combinations with other therapeutic agents, prodrugformulations are also provided in WO 2007/031091, which is herebyincorporated by reference. The invention also provides for apharmaceutical composition comprising a compound or a conjugate asherein described or a conjugate and a pharmaceutically acceptablediluent, carrier or adjuvant. WO 2007/031091 provides suitable andpreferred pharmaceutically acceptable diluents, carriers and adjuvants,which are hereby incorporated by reference.

In various embodiments, the invention described herein encompasses amethod of preventing or treating a disease comprising administering atherapeutically effective amount of an oligomer that modulates PIK3CA(and in some embodiments, beta-catenin) to a human in need of suchtherapy. The invention further encompasses the use of a short period ofadministration of an oligomer that modulates PIK3CA (and in someembodiments, beta-catenin) or conjugate thereof, rather than continuousadministration.

EMBODIMENTS

The following embodiments of the present invention may be used incombination with the other embodiments described herein.

1. An oligomer of between 10-50 nucleobases in length which comprises acontiguous nucleobase sequence of a total of between 10-50 nucleobases,wherein said contiguous nucleobase sequence is at least 80% homologousto a corresponding region of a nucleic acid which encodes a mammalianPIK3CA kinase.

2. The oligomer according to embodiment 1, wherein said oligomercomprises at least one LNA unit.

3. The oligomer according to embodiment 1 or 2, wherein the contiguousnucleobase sequence comprises no more than 3, such as no more than 2mismatches to the corresponding region of a nucleic acid which encodes amammalian PIK3CA kinase.

4. The oligomer according to embodiment 3, wherein said contiguousnucleobase sequence comprises a single mismatch to the correspondingregion of a nucleic acid which encodes a mammalian PIK3CA kinase,wherein, optionally, the single mismatch corresponds to a singlenucleotide point mutation which is associated with a cancer phenotype.

5. The oligomer according to embodiment 1 or 2, wherein said contiguousnucleobase sequence comprises no mismatches, (i.e. is complementary to)the corresponding region of a nucleic acid which encodes a mammalianPIK3CA kinase.

6. The oligomer according to any one of embodiments 1-5, wherein thenucleobase sequence of the oligomer consists of the contiguousnucleobase sequence.

7. The oligomer according to any one of embodiments 1-6, wherein thenucleic acid which encodes a mammalian PIK3CA kinase is the human PIK3CAkinase nucleotide sequence such as SEQ ID No 1, or a variant thereof,such as SEQ ID NO 1 which comprises a single point mutation at aposition selected from 1781, 1790 and 3297.

8. The oligomer according to any one of embodiments 1-7, wherein thecontiguous nucleobase sequence is complementary to a correspondingregion of both the human PIK3CA kinase nucleic acid sequence and anon-human mammalian PIK3CA kinase nucleic acid sequence, such as themouse PIK3CA kinase nucleic acid sequence.

9. The oligomer according to any one of embodiments 1 to 8, wherein thecontiguous nucleobase sequence comprises a contiguous subsequence of atleast 7, nucleobase residues which, when formed in a duplex with thecomplementary PIK3CA kinase target RNA is capable of recruiting RNaseH.

10. The oligomer according to embodiment 9, wherein the contiguousnucleobase sequence comprises of a contiguous subsequence of at least 8,at least 9 or at least 10 nucleobase residues which, when formed in aduplex with the complementary PIK3CA kinase target RNA is capable ofrecruiting RNaseH.

11. The oligomer according to any one of embodiments 9 or 10 whereinsaid contiguous subsequence is at least 9 or at least 10 nucleobases inlength, such as at least 12 nucleobases or at least 14 nucleobases inlength, such as 14, 15 or 16 nucleobases residues which, when formed ina duplex with the complementary PIK3CA kinase target RNA is capable ofrecruiting RNaseH.

12. The oligomer according to embodiment any one of embodiments 1-11wherein said oligomer is conjugated with one or more non-nucleobasecompounds.

13. The oligomer according to any one of embodiments 1-12, wherein saidoligomer has a length of between 10-22 nucleobases.

14. The oligomer according to any one of embodiments 1-13, wherein saidoligomer has a length of between 12-18 nucleobases.

15. The oligomer according to any one of embodiments 1-14, wherein saidoligomer has a length of 14, 15 or 16 nucleobases.

16. The oligomer according to any one of embodiments 1-15, wherein saidcontinuous nucleobase sequence corresponds to a contiguous nucleotidesequence present in a nucleic acid sequence selected from the groupconsisting of SEQ ID NO 110-124, or 149-160.

17. The oligomer according to any one of embodiments 1-16, wherein theoligomer or contiguous nucleobase sequence comprises, or is selectedfrom a corresponding nucleobase sequence present in a nucleotidesequence selected from the group consisting of SEQ ID NO 2-16.

18. The oligomer according to any one of embodiments 1-17, wherein saidcontiguous nucleobase sequence comprises at least one affinity enhancingnucleotide analogue.

19. The oligomer according to embodiment 18, wherein said contiguousnucleobase sequence comprises a total of 2, 3, 4, 5, 6, 7, 8, 9 or 10affinity enhancing nucleotide analogues, such as between 5 and 8affinity enhancing nucleotide analogues.

20. The oligomer according to any one of embodiments 1-19 whichcomprises at least one affinity enhancing nucleotide analogue, whereinthe remaining nucleobases are selected from the group consisting of DNAnucleotides and RNA nucleotides, preferably DNA nucleotides.

21. The oligomer according to any one of embodiments 1-20, wherein theoligomer comprises of a sequence of nucleobases of formula, in 5′ to 3′direction, A-B-C, and optionally of formula A-B-C-D, wherein:

-   -   A consists or comprises of at least one nucleotide analogue,        such as 1, 2, 3, 4, 5 or 6 nucleotide analogues, preferably        between 2-5 nucleotide analogues, preferably 2, 3 or 4        nucleotide analogues, most preferably 2, 3 or 4 consecutive        nucleotide analogues and;    -   B consists or comprises at least five consecutive nucleobases        which are capable of recruiting RNAseH (when formed in a duplex        with a complementary RNA molecule, such as the PIK3CAK mRNA        target), such as DNA nucleobases, such as 5, 6, 7, 8, 9, 10, 11        or 12 consecutive nucleobases which are capable of recruiting        RNAseH, or between 6-10, or between 7-9, such as 8 consecutive        nucleobases which are capable of recruiting RNAseH, and;    -   C consists or comprises of at least one nucleotide analogue,        such as 1, 2, 3, 4, 5, or 6 nucleotide analogues, preferably        between 2-5 nucleotide analogues, such as 2, 3 or 4 nucleotide        analogues, most preferably 2, 3 or 4 consecutive nucleotide        analogues, and;    -   D when present, consists or comprises, preferably consists, of        one or more DNA nucleotide, such as between 1-3 or 1-2 DNA        nucleotides.

22. The oligomer according to embodiment 21, wherein region A consistsor comprises of 2, 3 or 4 consecutive nucleotide analogues.

23. The oligomer according to any one of embodiments 21-22, whereinregion B consists or comprises of 7, 8, 9 or 10 consecutive DNAnucleotides or equivalent nucleobases which are capable of recruitingRNAseH when formed in a duplex with a complementary RNA, such as thePIK3CA kinase mRNA target.

24. The oligomer according to any one of embodiments 21-24, whereinregion C consists or comprises of 2, 3 or 4 consecutive nucleotideanalogues.

25. The oligomer according to any one of embodiments 21-24, whereinregion D consists, where present, of one or two DNA nucleotides.

26. The oligomer according to any one of embodiments 21-25, wherein:

-   -   A Consists or comprises of 3 contiguous nucleotide analogues;    -   B Consists or comprises of 7, 8, 9 or 10 contiguous DNA        nucleotides or equivalent nucleobases which are capable of        recruiting RNAseH when formed in a duplex with a complementary        RNA, such as the PIK3CA kinase mRNA target;    -   A Consists or comprises of 3 contiguous nucleotide analogues;    -   B Consists, where present, of one or two DNA nucleotides.

27. The oligomer according to embodiment 26, wherein the contiguousnucleobase sequence consists of 10, 11, 12, 13 or 14 nucleobases, andwherein;

-   -   A. Consists of 1, 2 or 3 contiguous nucleotide analogues;    -   B. Consists of 7, 8, or 9 consecutive DNA nucleotides or        equivalent nucleobases which are capable of recruiting RNAseH        when formed in a duplex with a complementary RNA, such as the        PIK3CA kinase mRNA target;    -   A Consists of 1, 2 or 3 contiguous nucleotide analogues;    -   B Consists, where present, of one DNA nucleotide.

28. The oligomer according to anyone of embodiments 21-27, wherein Bcomprises at least one LNA nucleobase which is in the alpha-Lconfiguration, such as alpha-L-oxy LNA.

29. The oligomer according to any one of embodiments 1-28, wherein thenucleotide analogue(s) are independently or collectively selected fromthe group consisting of: Locked Nucleic Acid (LNA) units; 2′-O-alkyl-RNAunits, 2′-OMe-RNA units, 2′-amino-DNA units, 2′-fluoro-DNA units, PNAunits, HNA units, and INA units.

30. The oligomer according to embodiment 29 wherein all the nucleotideanalogues(s) are LNA units.

31. The oligomer according to any one of embodiments 1-30, whichcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 LNA units such as between 2and 8 nucleotide LNA units.

32. The oligomer according to any one of the embodiments 29-31, whereinthe LNAs are independently selected from oxy-LNA, thio-LNA, andamino-LNA, in either of the beta-D and alpha-L configurations orcombinations thereof.

33. The oligomer according to embodiment 32, wherein the LNAs are allbeta-D-oxy-LNA.

34. The oligomer according to any one of embodiments 21-33, wherein thenucleotide analogues of regions A and C are beta-D-oxy-LNA.

35. The oligomer according to any one of embodiments 1-34, wherein atleast one of the nucleobases present in the oligomers a modifiednucleobase selected from the group consisting of 5-methylcytosine,isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil,6-aminopurine, 2-aminopurine, inosine, diaminopurine, and2-chloro-6-aminopurine.

36. The oligomer according to any one of embodiments 1-35, wherein saidoligomer hybridises with a corresponding mammalian PIK3CA kinase mRNAwith a T_(m) of at least 50° C.

37. The oligomer according to any one of embodiments 1-36, wherein saidoligomer hybridises with a corresponding mammalian PIK3CA kinase mRNAwith a T_(m) of no greater than 80° C.

38. The oligomer according to any one of embodiments 1-37, wherein theinternucleoside linkages are independently selected from the groupconsisting of: phosphodiester, phosphorothioate and boranophosphate.

39. The oligomer according to embodiment 38, wherein the oligomercomprises at least one phosphorothioate internucleoside linkage.

40. The oligomer according to embodiment 39, wherein the internucleosidelinkages adjacent to and/or between DNA or RNA units, or within region Bare phosphorothioate linkages.

41. The oligomer according to embodiment 39 or 40, wherein the linkagesbetween at least one pair of consecutive nucleotide analogues is aphosphodiester linkage.

42. The oligomer according to embodiment 39 or 40, wherein all thelinkages between consecutive nucleotide analogues are phosphodiesterlinkages.

43. The oligomer according to embodiment 38 wherein all theinternucleoside linkages are phosphorothioate linkages.

44. A conjugate comprising the oligomer according to any one of theembodiments 1-43 and at least one non-nucleotide or non-polynucleotidemoiety covalently attached to said compound.

45. A pharmaceutical composition comprising an oligomer as defined inany of embodiments 1-43 or a conjugate as defined in embodiment 44, anda pharmaceutically acceptable diluent, carrier, salt or adjuvant.

46. A pharmaceutical composition according to 45, wherein the oligomeris constituted as a pro-drug.

47. Use of an oligomer as defined in any one of the embodiments 1-43, ora conjugate as defined in embodiment 44, for the manufacture of amedicament for the treatment of a disease or disorder selected from thegroup consisting of hyperproliferative diseases such as cancer.

48. A method for treating a hyperproliferative disease such as cancer,said method comprising administering an oligomer as defined in one ofthe embodiments 1-43, or a conjugate as defined in embodiment 44, or apharmaceutical composition as defined in any one of the embodiments45-46, to a patient in need thereof.

49. A method of reducing or inhibiting the expression of PIK3CA kinasein a cell or a tissue, the method comprising the step of contacting saidcell or tissue with a compound as defined in one of the embodiments1-43, or a conjugate as defined in embodiment 44, or a pharmaceuticalcomposition as defined in any one of the embodiments 45-46, so thatexpression of PIK3CA kinase is reduce or inhibited.

EXAMPLES Example 1 Monomer Synthesis

The LNA monomer building blocks and derivatives were prepared followingpublished procedures and references cited therein—see WO07/031,081,which is incorporated herein in its entirety, and the references citedtherein.

Example 2 Oligonucleotide Synthesis

Oligonucleotides were synthesized according to the method described inWO07/031,081. Table 2 shows examples of antisense oligonucleotidesequences of the invention. Tables 3 and 4 show examples of antisenseoligonucleotides (oligomers) of the invention.

Example 3 Design of the Oligonucleotides

In accordance with the present invention, a series of differentoligonucleotides were designed to target different regions of humanPIK3CA mRNA (phosphoinositide-3-kinase, catalytic, alpha polypeptide).(GenBank Accession number NM_(—)006218, SEQ ID NO: 1).

SEQ ID NOS 2-16: are oligomer sequences designed to target humanwild-type PIK3CA mRNA (i.e., that are fully complementary to a targetregion of a wild-type PIK3CA mRNA).

TABLE 2 Antisense oligonucleotide sequences Length Target site SEQ ID NOSequence (5′-3′) (bases) NM_006218 SEQ ID NO: 2 GAGGCATTCTAAAGTC 16253-268 SEQ ID NO: 3 ATTCTTCCCTTTCTGC 16 386-401 SEQ ID NO: 4TAGACATACATTGCTC 16 642-657 SEQ ID NO: 5 TACTTGCCCTGATATT 16 891-906 SEQID NO: 6 CACATAAGGGTTCTCC 16 1247-1262 SEQ ID NO: 7 AGCCATTCATTCCACC 161302-1317 SEQ ID NO: 8 CAGTAACACCAATAGG 16 1529-1544 SEQ ID NO: 9AACTCCAACTCTAAGC 16 1572-1587 SEQ ID NO: 10 CAGACAGAAGCAATTT 161856-1871 SEQ ID NO: 11 TTATTGTGCATCTCAG 16 2175-2190 SEQ ID NO: 12GCAGAGGACATAATTC 16 2466-2481 SEQ ID NO: 13 GATGTCTGGGTTCTCC 162506-2521 SEQ ID NO: 14 TTCTTCTTGTGATCCA 16 2970-2985 SEQ ID NO: 15AAGAAATCCTGTGTCA 16 3024-3039 SEQ ID NO: 16 TCTCCTGAAACCTCTC 163089-3104 SEQ ID NO: 110 CACGGAGGCATTCTAAAGTCACTA 24 249-272 SEQ ID NO:111 AAAAATTCTTCCCTTTCTGCTTCT 24 382-405 SEQ ID NO: 112AGGATAGACATACATTGCTCTACT 24 638-661 SEQ ID NO: 113AATATACTTGCCCTGATATTCTAA 24 887-910 SEQ ID NO: 114TTGTCACATAAGGGTTCTCCTCCA 24 1243-1266 SEQ ID NO: 115ATTCAGCCATTCATTCCACCTGGG 24 1298-1321 SEQ ID NO: 116GATCCAGTAACACCAATAGGGTTC 24 1525-1548 SEQ ID NO: 117GTCAAACTCCAACTCTAAGCATGG 24 1568-1591 SEQ ID NO: 118TTAACAGACAGAAGCAATTTGGGT 24 1852-1875 SEQ ID NO: 119TGTTTTATTGTGCATCTCAGATTT 24 2171-2194 SEQ ID NO: 120TTTTGCAGAGGACATAATTCGACA 24 2462-2485 SEQ ID NO: 121ACATGATGTCTGGGTTCTCCCAAT 24 2502-2525 SEQ ID NO: 122TTTTTTCTTCTTGTGATCCAAAAA 24 2966-2989 SEQ ID NO: 123TATTAAGAAATCCTGTGTCAAAAC 24 3020-3043 SEQ ID NO: 124CACATCTCCTGAAACCTCTCAAAT 24 3085-3108

SEQ ID NOS: 17-22 show oligomer sequences designed to target the targetregions of variants of human PIK3CA mRNA comprising each of the threehot-spot mutations (E542K, E545K and 111047R), and SEQ ID NOS: 23-28show oligomer sequences that are fully complementary to the same targetregions (i.e., those comprising hot-spot mutations in PIK3CA variants)of human wild-type PIK3CA. The base of each oligomer (having SEQ ID NOs:23-28) that base pairs with the mutated base in the target regions ofthe variant PIK3CA nucleic acids is highlighted. The sequences of SEQ IDNOs: 125-136, respectively include the 16-mer sequences shown in SEQ IDNOs: 17-22 with additional monomers flanking the 16-mer sequences at the5′ and 3′ ends. SEQ ID NOs: 17, 18, 125, and 126 are targeted to thetarget region comprising the E542K mutation. SEQ ID NOs: 19, 20, 127 and128 are targeted to the target region comprising the E545K mutation, andSEQ ID NOs: 21, 22, 129 and 130 are targeted to the target regioncomprising the H1047R mutation.

TABLE 3 Antisense oligonucleotide sequences targeted to wild-type ormutant target regions of PIK3CA Length Target site SEQ ID NO Sequence(5′-3′) (bases) NM_006218 SEQ ID NO: 17 GATTTTAGAGAGAGGA 16 1771-1786SEQ ID NO: 18 AGTGATTTTAGAGAGA 16 1774-1789 SEQ ID NO: 19TCCTGCTTAGTGATTT 16 1782-1797 SEQ ID NO: 20 TTCTCCTGCTTAGTGA 161785-1800 SEQ ID NO: 21 GCCACCATGACGTGCA 16 3292-3307 SEQ ID NO: 22ACCATGACGTGCATCA 16 3289-3304 SEQ ID NO: 23 GATTTCAGAGAGAGGA 161771-1786 SEQ ID NO: 24 AGTGATTTCAGAGAGA 16 1774-1789 SEQ ID NO: 25TCCTGCTCAGTGATTT 16 1782-1797 SEQ ID NO: 26 TTCTCCTGCTCAGTGA 161785-1800 SEQ ID NO: 27 GCCACCATGATGTGCA 16 3292-3307 SEQ ID NO: 28ACCATGATGTGCATCA 16 3289-3304 SEQ ID NO: 125 CAGTGATTTTAGAGAGAGGATCTC 241767-1790 SEQ ID NO: 126 GCTCAGTGATTTTAGAGAGAGGAT 24 1770-1793 SEQ IDNO: 127 TTTCTCCTGCTTAGTGATTTCAGA 24 1778-1801 SEQ ID NO: 128ATCTTTCTCCTGCTTAGTGATTTC 24 1781-1804 SEQ ID NO: 129TCCAGCCACCATGACGTGCATCAT 24 3288-3311 SEQ ID NO: 130AGCCACCATGACGTGCATCATTCA 24 3285-3308 SEQ ID NO: 131CAGTGATTTCAGAGAGAGGATCTC 24 1767-1790 SEQ ID NO: 132GCTCAGTGATTTCAGAGAGAGGAT 24 1770-1793 SEQ ID NO: 133TTTCTCCTGCTCAGTGATTTCAGA 24 1778-1801 SEQ ID NO: 134ATCTTTCTCCTGCTCAGTGATTTC 24 1781-1804 SEQ ID NO: 135TCCAGCCACCATGATGTGCATCAT 24 3288-3311 SEQ ID NO: 136AGCCACCATGATGTGCATCATTCA 24 3285-3308

In SEQ ID NOs: 29-55, upper case letters indicates nucleoside analogueunits (nucleoside analogue monomers), such as LNA monomers, andsubscript “s” represents a phosphorothiote linkage. The absence of “s”(if any) indicates phosphodiester linkage. Lower case letters representnucleoside (such as DNA or RNA monomers). All cytosine bases in the LNAmonomers are 5-methylcytosines.

TABLE 4 Oligonucleotide designs of the invention SEQ ID NO Sequence(5′-3′) SEQ ID NO: 29 G _(s) A _(s) G_(s)g_(s)c_(s)a_(s)t_(s)t_(s)c_(s)t_(s)a_(s)a_(s)a_(s) G _(s) T _(s) CSEQ ID NO: 30 A _(s) T _(s) T_(s)c_(s)t_(s)t_(s)c_(s)c_(s)c_(s)t_(s)t_(s)t_(s)c_(s) T _(s) G _(s) CSEQ ID NO: 31 T _(s) A _(s) G_(s)a_(s)c_(s)a_(s)t_(s)a_(s)c_(s)a_(s)t_(s)t_(s)g_(s) C _(s) T _(s) CSEQ ID NO: 32 T _(s) A _(s) C_(s)t_(s)t_(s)g_(s)c_(s)c_(s)c_(s)t_(s)g_(s)a_(s)t_(s) A _(s) T _(s) TSEQ ID NO: 33 C _(s) A _(s) C_(s)a_(s)t_(s)a_(s)a_(s)g_(s)g_(s)g_(s)t_(s)t_(s)c_(s) T _(s) C _(s) CSEQ ID NO: 34 A _(s) G _(s) C_(s)c_(s)a_(s)t_(s)t_(s)c_(s)a_(s)t_(s)t_(s)c_(s)c_(s) A _(s) C _(s) CSEQ ID NO: 35 C _(s) A _(s) G_(s)t_(s)a_(s)a_(s)c_(s)a_(s)c_(s)c_(s)a_(s)a_(s)t_(s) A _(s) G _(s) GSEQ ID NO: 36 A _(s) A _(s) C_(s)t_(s)c_(s)c_(s)a_(s)a_(s)c_(s)t_(s)c_(s)t_(s)a_(s) A _(s) G _(s) CSEQ ID NO: 37 C _(s) A _(s) G_(s)a_(s)c_(s)a_(s)g_(s)a_(s)a_(s)g_(s)c_(s)a_(s)a_(s) T _(s) T _(s) TSEQ ID NO: 38 T _(s) T _(s) A_(s)t_(s)t_(s)g_(s)t_(s)g_(s)c_(s)a_(s)t_(s)c_(s)t_(s) C _(s) A _(s) GSEQ ID NO: 39 G _(s) C _(s) A_(s)g_(s)a_(s)g_(s)g_(s)a_(s)c_(s)a_(s)t_(s)a_(s)a_(s) T _(s) T _(s) CSEQ ID NO: 40 G _(s) A _(s) T_(s)g_(s)t_(s)c_(s)t_(s)g_(s)g_(s)g_(s)t_(s)t_(s)c_(s) T _(s) C _(s) CSEQ ID NO: 41 T _(s) T _(s) C_(s)t_(s)t_(s)c_(s)t_(s)t_(s)g_(s)t_(s)g_(s)a_(s)t_(s) C _(s) C _(s) ASEQ ID NO: 42 A _(s) A _(s) G_(s)a_(s)a_(s)a_(s)t_(s)c_(s)c_(s)t_(s)g_(s)t_(s)g_(s) T _(s) C _(s) ASEQ ID NO: 43 T _(s) C _(s) T_(s)c_(s)c_(s)t_(s)g_(s)a_(s)a_(s)a_(s)c_(s)c_(s)t_(s) C _(s) T _(s) CSEQ ID NO: 44 G _(s) A _(s) T_(s)t_(s)t_(s)t_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s)a_(s) G _(s) G _(s) ASEQ ID NO: 45 A _(s) G _(s) T_(s)g_(s)a_(s)t_(s)t_(s)t_(s)t_(s)a_(s)g_(s)a_(s)g_(s) A _(s) G _(s) ASEQ ID NO: 46 T _(s) C _(s) C_(s)t_(s)g_(s)c_(s)t_(s)t_(s)a_(s)g_(s)t_(s)g_(s)a_(s) T _(s) T _(s) TSEQ ID NO: 47 T _(s) T _(s) C_(s)t_(s)c_(s)c_(s)t_(s)g_(s)c_(s)t_(s)t_(s)a_(s)g_(s) T _(s) G _(s) ASEQ ID NO: 48 G _(s) C _(s) C_(s)a_(s)c_(s)c_(s)a_(s)t_(s)g_(s)a_(s)c_(s)g_(s)t_(s) G _(s) C _(s) ASEQ ID NO: 49 A _(s) C _(s) C_(s)a_(s)t_(s)g_(s)a_(s)c_(s)g_(s)t_(s)g_(s)c_(s)a_(s) T _(s) C _(s) ASEQ ID NO: 50 G _(s) A _(s) T_(s)t_(s)t_(s)c_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s)a_(s) G _(s) G _(s) ASEQ ID NO: 51 A _(s) G _(s) T_(s)g_(s)a_(s)t_(s)t_(s)t_(s)c_(s)a_(s)g_(s)a_(s)g_(s) A _(s) G _(s) ASEQ ID NO: 52 T _(s) C _(s) C_(s)t_(s)g_(s)c_(s)t_(s)c_(s)a_(s)g_(s)t_(s)g_(s)a_(s) T _(s) T _(s) TSEQ ID NO: 53 T _(s) T _(s) C_(s)t_(s)c_(s)c_(s)t_(s)g_(s)c_(s)t_(s)c_(s)a_(s)g_(s) T _(s) G _(s) ASEQ ID NO: 54 G _(s) C _(s) C_(s)a_(s)c_(s)c_(s)a_(s)t_(s)g_(s)a_(s)t_(s)g_(s)t_(s) G _(s) C _(s) ASEQ ID NO: 55 A _(s) C _(s) C_(s)a_(s)t_(s)g_(s)a_(s)t_(s)g_(s)t_(s)g_(s)c_(s)a_(s) T _(s) C _(s) A

Example 4 In Vitro Model: Cell Culture

The effect of antisense oligonucleotides on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. The target canbe expressed endogenously or by transient or stable transfection of anucleic acid encoding said target. The expression level of targetnucleic acid can be routinely determined using, for example, Northernblot analysis, Real-Time PCR, Ribonuclease protection assays. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen.

Cells were cultured in the appropriate medium as described below andmaintained at 37° C. at 95-98% humidity and 5% CO₂. Cells were routinelypassaged 2-3 times weekly.

MCF7: The human breast adenocarcinoma cell line MCF7 was cultured inEagle MEM (Sigma)+10% fetal bovine serum (FBS)+2 mM GlutamaxI+gentamicin (25 μg/ml)+1× Non Essential Amino Acid.

PC3: The human prostate adenocarcinoma cell line PC3 was cultured inDMEM (Sigma)+10% fetal bovine serum (FBS)+2 mM Glutamax I+gentamicin (25μg/ml).

Example 5 In Vitro Model: Treatment with Antisense Oligonucleotide

The cell lines listed in Example 4 were treated with oligonucleotideusing the cationic liposome formulation LipofectAMINE 2000 (Gibco) astransfection vehicle. Cells were seeded in 6-well cell culture plates(NUNC) and treated when 80-90% confluent. Oligomer concentrations usedranged from 0.8 nM to 20 nM final concentration. Formulation ofoligomer-lipid complexes were carried out essentially as described bythe manufacturer using serum-free OptiMEM (Gibco) and a final lipidconcentration of 2.5 μg/mL (PC3) or 5 μg/mL (MCF7) LipofectAMINE 2000.Cells were incubated at 37° C. for 4 hours and treatment was stopped byremoval of oligomer-containing culture medium. Cells were washed andserum-containing media was added. After oligomer treatment cells, wereallowed to recover for 20 hours before they were harvested for RNAanalysis.

Example 6 In Vitro Model: Extraction of RNA and cDNA Synthesis

Total RNA Isolation and First strand synthesis: Total RNA was extractedfrom cells transfected as described above and using the Qiagen RNeasykit (Qiagen cat. no. 74104) according to the manufacturer'sinstructions. First strand synthesis was performed using ReverseTranscriptase reagents from Ambion according to the manufacturer'sinstructions.

For each sample 0.5 •g total RNA was adjusted to (10.8 •l) with RNasefree H₂O and mixed with 2 •l random decamers (50 •M) and 4 •l dNTP mix(2.5 mM each dNTP) and heated to 70° C. for 3 min after which thesamples were rapidly cooled on ice. After cooling the samples on ice, 2•l 10× Buffer RT, 1 •l MMLV Reverse Transcriptase (100 U/•l) and 0.25 •lRNase inhibitor (10 U/•l) were added to each sample, followed byincubation at 42° C. for 60 min, heat inactivation of the enzyme at 95°C. for 10 min and then the sample was cooled to 4° C.

Example 7 In Vitro Model: Analysis of Oligonucleotide Inhibition ofPIK3CA Expression by Real-time PCR

Antisense modulation of PIK3CA expression can be assayed in a variety ofways known in the art. For example, PIK3CA mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR. Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor mRNA.

Methods of RNA isolation and RNA analysis such as Northern blotanalysis, are routine in the art and are taught in, for example, CurrentProtocols in Molecular Biology, John Wiley and Sons.

Real-time quantitative PCR can be conveniently accomplished using thecommercially available Multi-Color Real Time PCR Detection System,available from Applied Biosystems.

Real-time Quantitative PCR Analysis of PIK3CA mRNA Levels: The samplecontent of human PIK3CA mRNA was quantified using the human PIK3CA ABIPrism Pre-Developed TaqMan Assay Reagents (Applied Biosystems cat. no.Hs00180679_ml) according to the manufacturer's instructions.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA quantity was usedas an endogenous control for normalizing any variance in samplepreparation.

The sample content of human GAPDH mRNA was quantified using the humanGAPDH ABI Prism Pre-Developed TaqMan Assay Reagent (Applied Biosystemscat. no. 4310894E) according to the manufacturer's instructions.

Real-time Quantitative PCR is a technique well known in the art and istaught in for example Heid et al. Real time quantitative PCR, GenomeResearch (1996), 6: 986-994.

Real time PCR: The cDNA from the first strand synthesis performed asdescribed in Example 5 was diluted 2-20 times, and analyzed by real timequantitative PCR using Taqman 7500 FAST or 7900 FAST from AppliedBiosystems. The primers and probe were mixed with 2× Taqman FastUniversal PCR master mix (2×) (Applied Biosystems Cat.# 4364103) andadded to 4 μl cDNA to a final volume of 10 μl. Each sample was analysedin duplicate. Assaying 2-fold dilutions of a cDNA that had been preparedon material purified from a cell line expressing the RNA of interestgenerated standard curves for the assays. Sterile 1120 was used insteadof cDNA for the no-template control. PCR program: 95° C. for 30 seconds,followed by 40 cycles of 95° C., 3 seconds, 60° C., 20-30 seconds.Relative quantities of target mRNA sequence were determined from thecalculated Threshold cycle using the Applied Biosystems Fast System SDSSoftware Version 1.3.1.21. or SDS Software Version 2.3.

Example 8 In Vitro Analysis: Antisense Inhibition of Human PIK3CA mRNAExpression by Oligonucleotide Compounds

Oligonucleotides presented in Table 4 were evaluated for their potentialto knock down expression of human PIK3CA mRNA at concentrations of 0.8nM, 4 nM and 20 nM in PC3 cells and MCF7 cells (see FIGS. 1 and 2). Themost active oligonucleotides, having SEQ ID NOs: 57, 60, 64, 67, 77 and82 were further evaluated for their ability to knock down human PIK3CAmRNA at the concentrations 0.04 nM, 0.2 nM, 0.8 nM, 4 nM, 10 nM and 20nM in PC3 and MCF7 cells (see FIGS. 22 and 23).

The data in Table 5 are presented as percentage down-regulation relativeto mock transfected cells at 20 nM. Lower case letters represent DNAmonomers, bold upper case letters represent β-D-oxy-LNA monomers. Allcytosines in LNA monomers are 5-methylcytosines. Subscript “s”represents a phosphorothioate linkage.

TABLE 5 Antisense Inhibition of Human PIK3CA mRNA expression byoligonucleotides Percent Percent inhibition of inhibition of PIK3CA inPIK3CA in Test substance Sequence (5′-3′) MCF7 PC3 SEQ ID NO: 56 G _(s)A _(s) G _(s)g_(s)c_(s)a_(s)t_(s)t_(s)c_(s)t_(s)a_(s)a_(s)a_(s) G _(s) T_(s) C 47 48 SEQ ID NO: 57 A _(s) T _(s) T_(s)c_(s)t_(s)t_(s)c_(s)c_(s)c_(s)t_(s)t_(s)t_(s)c_(s) T _(s) G _(s) C94 91 SEQ ID NO: 58 T _(s) T _(s) C_(s)t_(s)t_(s)c_(s)c_(s)c_(s)t_(s)t_(s)t_(s)c_(s) T _(s) G n.d. n.d. SEQID NO: 59 T _(s) C _(s) t _(s)t_(s)c_(s)c_(s)c_(s)t_(s)t_(s)t_(s) C _(s)T n.d. n.d. SEQ ID NO: 60 T _(s) A _(s) G_(s)a_(s)c_(s)a_(s)t_(s)a_(s)c_(s)a_(s)t_(s)t_(s)g_(s) C _(s) T _(s) C96 96 SEQ ID NO: 61 A _(s) G _(s) A_(s)c_(s)a_(s)t_(s)a_(s)c_(s)a_(s)t_(s)t_(s)g_(s) C _(s) T n.d. n.d. SEQID NO: 62 G _(s) A _(s)c_(s)a_(s)t_(s)a_(s)c_(s)a_(s)t_(s)t_(s) G _(s) Cn.d. n.d. SEQ ID NO: 63 T _(s) A _(s) C_(s)t_(s)t_(s)g_(s)c_(s)c_(s)c_(s)t_(s)g_(s)a_(s)t_(s) A _(s) T _(s) T94 89 SEQ ID NO: 64 C _(s) A _(s) C_(s)a_(s)t_(s)a_(s)a_(s)g_(s)g_(s)g_(s)t_(s)t_(s)c_(s) T _(s) C _(s) C97 95 SEQ ID NO: 65 A _(s) C _(s) A_(s)t_(s)a_(s)a_(s)g_(s)g_(s)g_(s)t_(s)t_(s)c_(s) T _(s) C n.d. n.d. SEQID NO: 66 C _(s) A _(s)t_(s)a_(s)a_(s)g_(s)g_(s)g_(s)t_(s)t_(s) C _(s) Tn.d. n.d. SEQ ID NO: 67 A _(s) G _(s) C_(s)c_(s)a_(s)t_(s)t_(s)c_(s)a_(s)t_(s)t_(s)c_(s)c_(s) A _(s) C _(s) C97 93 SEQ ID NO: 68 G _(s) C _(s) C_(s)a_(s)t_(s)t_(s)c_(s)a_(s)t_(s)t_(s)c_(s)c_(s) A _(s) C n.d. n.d. SEQID NO: 69 C _(s) C _(s)a_(s)t_(s)t_(s)c_(s)a_(s)t_(s)t_(s)c_(s) C _(s) An.d. n.d. SEQ ID NO: 70 C _(s) A _(s) G_(s)t_(s)a_(s)a_(s)c_(s)a_(s)c_(s)c_(s)a_(s)a_(s)t_(s) A _(s) G _(s) G92 94 SEQ ID NO: 71 A _(s) G _(s) T_(s)a_(s)a_(s)c_(s)a_(s)c_(s)c_(s)a_(s)a_(s)t_(s) A _(s) G n.d. n.d. SEQID NO: 72 G _(s) T _(s)a_(s)a_(s)c_(s)a_(s)c_(s)c_(s)a_(s)a_(s) T _(s) An.d. n.d. SEQ ID NO: 73 A _(s) A _(s) C_(s)t_(s)c_(s)c_(s)a_(s)a_(s)c_(s)t_(s)c_(s)t_(s)a_(s) A _(s) G _(s) C81 75 SEQ ID NO: 74 C _(s) A _(s) G_(s)a_(s)c_(s)a_(s)g_(s)a_(s)a_(s)g_(s)c_(s)a_(s)a_(s) T _(s) T _(s) T90 94 SEQ ID NO: 75 A _(s) G _(s) A_(s)c_(s)a_(s)g_(s)a_(s)a_(s)g_(s)c_(s)a_(s)a_(s) T _(s) T n.d. n.d. SEQID NO: 76 G _(s) A _(s)c_(s)a_(s)g_(s)a_(s)a_(s)g_(s)c_(s)a_(s) A _(s) Tn.d. n.d. SEQ ID NO: 77 T _(s) T _(s) A_(s)t_(s)t_(s)g_(s)t_(s)g_(s)c_(s)a_(s)t_(s)c_(s)t_(s) C _(s) A _(s) G91 96 SEQ ID NO: 78 T _(s) A _(s) T_(s)t_(s)g_(s)t_(s)g_(s)c_(s)a_(s)t_(s)c_(s)t_(s) C _(s) A n.d. n.d. SEQID NO: 79 A _(s) T _(s)t_(s)g_(s)t_(s)g_(s)c_(s)a_(s)t_(s)c_(s) T _(s) Cn.d. n.d. SEQ ID NO: 80 G _(s) C _(s) A_(s)g_(s)a_(s)g_(s)g_(s)a_(s)c_(s)a_(s)t_(s)a_(s)a_(s) T _(s) T _(s) C76 80 SEQ ID NO: 81 G _(s) A _(s) T_(s)g_(s)t_(s)c_(s)t_(s)g_(s)g_(s)g_(s)t_(s)t_(s)c_(s) T _(s) C _(s) C90 81 SEQ ID NO: 82 T _(s) T _(s) C_(s)t_(s)t_(s)c_(s)t_(s)t_(s)g_(s)t_(s)g_(s)a_(s)t_(s) C _(s) C _(s) A92 93 SEQ ID NO: 83 T _(s) C _(s) T_(s)t_(s)c_(s)t_(s)t_(s)g_(s)t_(s)g_(s)a_(s)t_(s) C _(s) C n.d. n.d. SEQID NO: 84 C _(s) T _(s)t_(s)c_(s)t_(s)t_(s)g_(s)t_(s)g_(s)a_(s) T _(s) Cn.d. n.d. SEQ ID NO: 85 A _(s) A _(s) G_(s)a_(s)a_(s)a_(s)t_(s)c_(s)c_(s)t_(s)g_(s)t_(s)g_(s) T _(s) C _(s) A87 88 SEQ ID NO: 86 T _(s) C _(s) T_(s)c_(s)c_(s)t_(s)g_(s)a_(s)a_(s)a_(s)c_(s)c_(s)t_(s) C _(s) T _(s) C85 83 SEQ ID NO: 87 G _(s) A _(s) T_(s)t_(s)t_(s)c_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s)a_(s) G _(s) G _(s) A94 94 SEQ ID NO: 88 A _(s) T _(s) T_(s)t_(s)c_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s)a_(s) G _(s) G n.d. n.d. SEQID NO: 89 T _(s) T _(s)t_(s)c_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s) A _(s) Gn.d. n.d. SEQ ID NO: 90 A _(s) G _(s) T_(s)g_(s)a_(s)t_(s)t_(s)t_(s)c_(s)a_(s)g_(s)a_(s)g_(s) A _(s) G _(s) A96 92 SEQ ID NO: 91 G _(s) T _(s) G_(s)a_(s)t_(s)t_(s)t_(s)c_(s)a_(s)g_(s)a_(s)g_(s) A _(s) G n.d. n.d. SEQID NO: 92 T _(s) G _(s)a_(s)t_(s)t_(s)t_(s)c_(s)a_(s)g_(s)a_(s) G _(s) An.d. n.d. SEQ ID NO: 93 T _(s) C _(s) C_(s)t_(s)g_(s)c_(s)t_(s)c_(s)a_(s)g_(s)t_(s)g_(s)a_(s) T _(s) T _(s) T85 86 SEQ ID NO: 94 T _(s) T _(s) C_(s)t_(s)c_(s)c_(s)t_(s)g_(s)c_(s)t_(s)c_(s)a_(s)g_(s) T _(s) G _(s) A78 83 SEQ ID NO: 95 G _(s) C _(s) C_(s)a_(s)c_(s)c_(s)a_(s)t_(s)g_(s)a_(s)t_(s)g_(s)t_(s) G _(s) C _(s) A84 75 SEQ ID NO: 96 A _(s) C _(s) C_(s)a_(s)t_(s)g_(s)a_(s)t_(s)g_(s)t_(s)g_(s)c_(s)a_(s) T _(s) C _(s) A94 91 SEQ ID NO: 97 C _(s) C _(s) A _(s)_(s)g_(s)a_(s)t_(s)g_(s)t_(s)g_(s)c_(s)a_(s) T _(s) C n.d. n.d. SEQ IDNO: 98 C _(s) A _(s)t_(s)g_(s)a_(s)t_(s)g_(s)t_(s)g_(s)c_(s) A _(s) Tn.d. n.d. SEQ ID NO: 99 G _(s) A _(s) T_(s)t_(s)t_(s)t_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s)a_(s) G _(s) G _(s) An.d. n.d. SEQ ID NO: 137 A _(s) T _(s) T_(s)t_(s)t_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s) A _(s) G _(s) G n.d. n.d.SEQ ID NO: 138 T _(s) T _(s)t_(s)t_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s) A_(s) G n.d. n.d. SEQ ID NO: 100 A _(s) G _(s) T_(s)g_(s)a_(s)t_(s)t_(s)t_(s)t_(s)a_(s)g_(s)a_(s)g_(s) A _(s) G _(s) An.d. n.d. SEQ ID NO: 139 G _(s) T _(s) G_(s)a_(s)t_(s)t_(s)t_(s)t_(s)a_(s)g_(s)a_(s) G _(s) A _(s) G n.d. n.d.SEQ ID NO: 140 T _(s) G _(s)a_(s)t_(s)t_(s)t_(s)t_(s)a_(s)g_(s)a_(s) G_(s) A n.d. n.d. SEQ ID NO: 101 T _(s) C _(s) C_(s)t_(s)g_(s)c_(s)t_(s)t_(s)a_(s)g_(s)t_(s)g_(s)a_(s) T _(s) T _(s) Tn.d. n.d. SEQ ID NO: 141 C _(s) C _(s) T_(s)g_(s)c_(s)t_(s)t_(s)a_(s)g_(s)t_(s)g_(s) A _(s) T _(s) T n.d. n.d.SEQ ID NO: 142 C _(s) T _(s)g_(s)c_(s)t_(s)t_(s)a_(s)g_(s)t_(s)g_(s) A_(s) T n.d. n.d. SEQ ID NO: 102 T _(s) T _(s) C_(s)t_(s)c_(s)c_(s)t_(s)g_(s)c_(s)t_(s)t_(s)a_(s)g_(s) T _(s) G _(s) An.d. n.d. SEQ ID NO: 143 T _(s) C _(s) T_(s)c_(s)c_(s)t_(s)g_(s)c_(s)t_(s)t_(s)a_(s) G _(s) T _(s) G n.d. n.d.SEQ ID NO: 144 C _(s) T _(s)c_(s)c_(s)t_(s)g_(s)c_(s)t_(s)t_(s)a_(s) G_(s) T n.d. n.d. SEQ ID NO: 103 G _(s) C _(s) C_(s)a_(s)c_(s)c_(s)a_(s)t_(s)g_(s)a_(s)c_(s)g_(s)t_(s) G _(s) C _(s) An.d. n.d. SEQ ID NO: 145 C _(s) C _(s) A_(s)c_(s)c_(s)a_(s)t_(s)g_(s)a_(s)c_(s)g_(s) T _(s) G _(s) C n.d. n.d.SEQ ID NO: 146 C _(s) A _(s)c_(s)c_(s)a_(s)t_(s)g_(s)a_(s)c_(s)g_(s) T_(s) G n.d. n.d. SEQ ID NO: 104 A _(s) C _(s) C_(s)a_(s)t_(s)g_(s)a_(s)c_(s)g_(s)t_(s)g_(s)c_(s)a_(s) T _(s) C _(s) An.d. n.d. SEQ ID NO: 147 C _(s) C _(s) A_(s)t_(s)g_(s)a_(s)c_(s)g_(s)t_(s)g_(s)c_(s) A _(s) T _(s) C n.d. n.d.SEQ ID NO: 148 C _(s) A _(s)t_(s)g_(s)a_(s)c_(s)g_(s)t_(s)g_(s)c_(s) A_(s) T n.d. n.d.

As shown in Table 5, oligonucleotides having the sequences of SEQ IDNOs: 57, 60, 64, 67, 70, 74, 77, 82, 87, 90 and 96 demonstrated about90% or greater inhibition of PIK3CA mRNA expression at 20 nM in PC3 andMCF7 cells in these experiments.

In certain embodiments, oligomers based on the tested antisense oligomersequences and designs, but having, for example, different lengths(shorter or longer) and/or monomer content (e.g. the type and/or numberof nucleoside analogues) than those shown, e.g., in Table 5, could alsoprovide suitable inhibition of PIK3CA expression.

Example 9 In Vitro Analysis: Effect of Antisense Inhibition of HumanPIK3CA mRNA on Cell Proliferation (MTS Assay)

MCF7 breast cancer, PC3 prostate cancer and HCT116 colon cancer cellswere treated with oligonucleotides using the cationic liposomeformulation LipofectAMINE 2000 (Invitrogen) as transfection vehicle.Cells were seeded in 6-well culture plates (NUNC) the day beforetransfection at a density of 2.5×10⁵ cells/well (MCF7 and HCT116) or at2.4×10⁵ cells/well (PC3). The cells were treated when 75-90% confluentwith different concentrations of oligomers. Formulation ofoligomer-lipid complexes was carried out using serum-free OptiMEM(Invitrogen) and a final lipid concentration of 2.5 μg/ml (PC3), 5 μg/ml(MCF7) or 10 μg/ml (HCT116) LipofectAMINE 2000. Cells were incubated at37° C. for 4 hours and transfection was stopped by removal ofoligomer-containing culture medium. After 4 hours of treatment, mediawas removed and cells were trypsinized and seeded to a density of 5000cells per well in clear 96 well plate (Scientific Orange no. 1472030100)in 100 μl media. Viable cells were measured at the times indicated byadding 10 μl of the tetrazolium compound[3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] and an electron coupling reagent (phenazineethosulfate; PES) (CellTiter 96® AQueous One Solution Cell ProliferationAssay, Promega). Viable cells were measured at 490 nm in a Powerwave(Biotek Instruments). The OD490 nm was plotted against time/h. (SeeFIGS. 11, 12 and 14). As shown in FIG. 11, oligonucleotides having thesequences of SEQ ID NOs: 57, 60, 64, 67, 77 and 82 inhibitedproliferation of the breast cancer cell line MCF7, while none of theoligonucleotides had any pronounced effect on proliferation of PC3 cells(FIG. 12). Oligonucleotides having the sequences of SEQ ID NOs: 57, 60,67, 77 and 82 inhibited the proliferation of HCT116 cells. The PIK3CAgene in the cell line MCF7 has the E545K hot-spot mutation and thePIK3CA gene in the HCT116 cell line has the H1047R hot-spot mutation.These hot-spot mutations are activating mutations. As a result, MCF7 andHCT116 cells are more sensitive to changes in PIK3CA signalling than thePC3 cell line, which has no reported hot-spot mutation in the PIK3CAgene.

Example 10 In Vitro Analysis: Effect on Caspase-3/7 Induction afterAntisense Inhibition of PIK3CA in Human Cancer Cell Lines

PC3 prostate cancer and HCT116 colon cancer cells were treated witholigonucleotides using the cationic liposome formulation LipofectAMINE2000 (Invitrogen) as transfection vehicle. Cells were seeded in 6-wellculture plates (NUNC) the day before transfection at a density of2.5×10⁵ cells/well (MCF7 and HCT116) or of 2.4×10⁵ cells/well (PC3). Thecells were treated when 75-90% confluent with different concentrationsof oligomers. Formulation of oligomer-lipid complexes was carried outusing serum-free OptiMEM (Invitrogen) and a final lipid concentration of2.5 μg/ml (PC3) or 10 μg/ml (HCT116) LipofectAMINE 2000. Cells wereincubated at 37° C. for 4 hours and transfection was stopped by removalof oligomer-containing culture medium.

After 4 hours of treatment, media was removed and cells were trypsinizedand seeded to a density of 5000 cells per well in white 96 well plate(Nunc) in 100 μl media. Caspase 3/7 activity was measured at the timesindicated by adding 100 μl Caspase-Glo 3/7 assay (Promega). Caspase 3/7activity was measured using a luminometer. The caspase 3/7 activitieswere measured at four different time points 24 h, 32 h, 48 h and 72 h(See FIG. 13 and FIG. 15). The oligomers having the sequences of SEQ IDNOs: 57, 67, 77 and 82 showed dose-dependent induction in Caspase 3/7activity in HCT116 cells, which has a PIK3CA gene have the H1047Rhot-spot mutation (FIG. 15), while the oligonucleotides had no effect oncaspase 3/7 induction in PC3 cells (which have no reported hot-spotmutation in the PIK3CA gene).

Example 11 In Vitro Analysis: Biostability of PIK3CA Oligonucleotides inMouse Plasma

Mouse plasma (Lithium heparin plasma from BomTac:NMRI mice, collected14-09-05, Taconic Europe) was defrosted and aliquoted into tubes with 45•l plasma/tube. Following this step, 5 •l oligomer (200 •M) was added tothe 45 •l plasma to a final concentration of 20 •M. After thoroughmixing, the samples were incubated at 37° C. for 0-120 hrs. At differenttime points (0 h, 24 h, 48 h and 120 h) samples were collected and thereaction was quenched by snap freezing the samples in liquid nitrogen.For analysis, loading buffer was added to the samples, which ere thenanalysed by electrophoresis on a PAGE-sequencing gel under denaturingconditions. The oligonucleotides of the invention showed high plasmastability for up to 120 hrs, as shown in FIG. 16.

Example 12 In Vitro Analysis: Tm Measurement of PIK3CA OligonucleotidesAgainst Complementary RNA

The melting temperature of antisense oligomer/RNA duplexes wasdetermined using a UV-spectrometry system with corresponding software(Perkin Elmer, Fremont, USA). The LNA oligomer and RNA comprising afully-complementary target region were added in final concentrations of1.5 •M to the T_(m)-buffer (200 nM NaCl, 0.2 nM EDTA, 20 mM NaP, pH7.0). Duplex formation was effected by heating the samples to 95° C. for3 min followed by cooling at room temperature for 30 min.

Melting temperature (T_(m)) values were measured in a Lambda 25 UV/VISspectrometer (Perkin Elmer) and data were collected and analysed usingthe TempLab software (Perkin Elmer). The instrument was programmed toheat the oligomer/RNA duplex sample from 20-95° C. and afterwardscooling the sample to 25° C. During this process the absorbance at 260nm was recorded. The melting curves were used to calculate T_(m) values.

The Tm values for SEQ ID NO: 57, 60, 64, 67, 77 and 82 are presented inFIG. 17.

Example 13 In Vitro Analysis: Down-Regulation of PIK3CA and pAkt in A549Cells

A549 cells were transfected with 30 nM of LNA and cultured for 24 h. WB:24 h, 20 ug protein/lane, 8% gel. Significant reduction of PIK3CA levelwas seen after transfection of A549 cells with PIK3CA LNAs. (FIG. 18)Reduction of pAk-t levels was also observed 24 h post transfection.(FIG. 18). Transfection with the oligomer having the sequence of SEQ IDNO: 60 had less effect on downstream signals compared to the othertested LNAs.

Example 14 In Vitro Analysis: Down-regulation of PIK3CA and pAkt in15PC3 Cells

15PC3 cell were transfected with 30 nM of LNA or treated with LY294200(a small molecule PI3K inhibitor) for 24 and 48 h. WB: 48 h, 15 ugprotein/lane, 8% gel. Significant reduction of PIK3CA levels wasobserved after transfection of 15PC3 cells with PIK3CA LNAs. (FIG. 19)pAkt levels were reduced up to 80% at 24 h post transfection determinedby ELISA. (FIG. 19). Transfection with the oligomer having the sequenceof SEQ ID NO: 82 had less effect on downstream signals compared to theother tested LNAs.

Example 15 In Vivo Analysis: Down-regulation of Mouse PIK3CA in MouseLiver after i.v. Administration of PIK3CA Oligonucleotides

Female NMRI mice received i.v. injection of oligonucleotides having thesequences of SEQ ID NO: 57, 60, 67, 77 and 82 on three consecutive daysat a dosage of 25 mg/kg. Animals were sacrificed 24 h after last dosing.The liver was stored in RNAlater stabilizing solution until use. TotalRNA was extracted from liver tissue and PIK3CA mRNA levels were analyzedwith qPCR. Data were compared to PIK3CA expression in saline treatedcontrol animals. As shown in FIG. 21, all oligonucleotides in the studyshowed potent down-regulation of PIK3CA mRNA.

All PIK3CA oligomers used in the second screening showed potentdown-regulation of PIK3CA in PC3 and MCF7 cells with IC₅₀ values below 1nM. SEQ ID NO: 82 also shows down-regulation of the control targetbeta-catenin gene in both MCF7 and PC3 cells (SEQ ID NO: 82 has 2mismatches when compared to the reverse complement of the best-alignedtarget region of beta-catenin).

Example 16 In Vivo Analysis: Effect of Oligomers on Lung Cancer TumorSize in Mice

Six-to seven-week old male athymic nu/nu mice (Harlan Sprague Dawley)weighing an average of 27.3±2.4 g were used in the study. Five millioncells of Calu-6 (lung cancer cell line) were suspended in PBS(Gibco#14190) were injected subcutaneously into each mouse. The micewere injected with two hundred μl of oligomer intravenously when theaverage tumor size reached 150 mm³. Oligomers were given every 3 daysfor a total of 5 dosings. The control vehicles were given the samedosing regimen as the oligomers. The tumor volumes for each mouse weredetermined by measuring two dimensions with calipers and calculatedusing the formula: tumor volume=(length×width²)/2). FIG. 20 shows downregulation of PIK3CA expression in liver by oligomers of the invention.

The following oligomers were found to have a good toxicity profile interms of good animal survival in in vivo experiments at 3 mg/kg dose—SEQIDs 60, 67, 77 & 82. SEQ IDs 60, 67 and 77 showed a good toxicityprofile at 10 mg/kg dosage.

TABLE 6 Effect of oligomers on PIK3CA mRNA in mouse liver and tumor andon tumor size Dose TGI on Group (mg/kg) Tumor KD Liver KD day 12 (%)Control — ND — SEQ ID 57 3 ND 20 ± 23 22.9 10 ND 62 ± 16 18.0 SEQ ID 603 ND 27 ± 14 24.7 10 ND 66 ± 11 53.6 30 ND 84 ± 8 49.4 100 ND 88 ± 4.054.1 SEQ ID 67 3 16 ± 12 33 ± 37 50.6 10 36 ± 11 73 ± 16 50.5 30 58 ±5.2 82 ± 17 44.5 100 65 ± 12 81 ± 4.2 31.5 SEQ ID 77 3 ND 65 ± 18 57.610 ND 86 ± 5.6 21.2 30 ND 91 ± 6.9 48.5 SEQ ID 82 3 ND 62 ± 26 37.5 10ND 72 ± 10 50.0

Example 17 Preparation of Conjugates of Oligomers with PolyethyleneGlycol

The oligomers having sequences shown as SEQ ID NO: 60 (IA) or SEQ ID NO:87 (IB) are functionalized on the 5′ terminus by attaching an aminoalkylgroup, such as hexan-1-amine blocked with a blocking group such as Fmocto the 5′ phosphate groups of the oligomers using routinephosphoramidite chemistry, oxidizing the resultant compounds,deprotecting them and purifying them to achieve the functionalizedoligomers, respectively, having the formulas (IA) and (IB):

wherein the bold uppercase letters represent nucleoside analoguemonomers, lowercase letters represent DNA monomers, the subscript “s”represents a phosphorothioate linkage, and ^(Me)C represents5-methylcytosine.

A solution of activated PEG, such as the one shown in formula (II):

wherein the PEG moiety has an average molecular weight of 12,000, andeach of the compounds of formulas (IA) and (IB) in PBS buffer arestirred in separate vessels at room temperature for 12 hours. Thereaction solutions are extracted three times with methylene chloride andthe combined organic layers are dried over magnesium sulphate andfiltered and the solvent is evaporated under reduced pressure. Theresulting residues are dissolved in double distilled water and loadedonto an anion exchange column. Unreacted PEG linker is eluted with waterand the products are eluted with NH₄HCO₃ solution. Fractions containingpure products are pooled and lypophilized to yield the conjugates SEQ IDNOs: 60 and 87, respectively as show in formulas (IIIA) and (IIIB):

wherein each of the oligomers of SEQ ID NOs: 60 and 87 is attached to aPEG polymer having average molecular weight of 12,000 via a releasablelinker.

Chemical structures of PEG polymer conjugates that can be made witholigomers having sequences shown in SEQ ID NOs: 67, 77 and 82 using theprocess described above are respectively shown in formulas (IVA), IVB)and (IVC):

wherein bold uppercase letters represent beta-D-oxy-LNA monomers,lowercase letters represent DNA monomers, the subscript “s” represents aphosphorothioate linkage and ^(Me)C represent 5-methylcytosine.

Activated oligomers that can be used in this process to respectivelymake the conjugates shown in formulas (IVA) (SEQ ID NO: 67), (IVB) (SEQID NO: 77) and (IVC) (SEQ ID NO: 82) have the chemical structures shownin formulas (VA),(VB) and (VC):

The invention claimed is:
 1. An oligomer, 5′-A_(s)G_(s)^(Me)C_(s)c_(s)a_(s)t_(s)t_(s)c_(s)a_(s)t_(s)t_(s)c_(s)c_(s)A_(s)^(Me)C_(s) ^(Me)C-3′ (SEQ ID NO: 67), wherein uppercase letters denotebeta-D-oxy-LNA monomers and lowercase letters denote DNA monomers, thesubscript “s” denotes a phosphorothioate linkage, and ^(Me)C denotes abeta-D-oxy-LNA monomer containing a 5-methylcytosine base.
 2. Apharmaceutical composition comprising the oligomer of claim 1, and apharmaceutically acceptable diluent, carrier, salt or adjuvant.
 3. Aconjugate comprising the oligomer of claim 1 covalently attached to atleast one moiety that is not a nucleic acid or a monomer.
 4. Apharmaceutical composition comprising the conjugate according to claim3, and a pharmaceutically acceptable diluent, carrier, salt or adjuvant.5. An activated oligomer consisting of the oligomer of claim 1 with atleast one functional group covalently attached thereto at one or morepositions independently selected from the 5′-end, the 3′ end, the 2′—OHof a ribose sugar, and the base.